- Email: [email protected]
Identification of Differential Patterns of Oxidative Biomarkers in Prostate Cancer Progression
Original Study
Identification of Differential Patterns of Oxidative Biomarkers in Prostate Cancer Progression Tianying Wu,1,2 Susan Kasper,3 Ronnie Meiyi Wong,1 Bruce Bracken4 Abstract This article focuses on prostate cancer (PCa) progression. The study population consisted of patients recruited from urologic clinics at the University of Cincinnati Medical Center. Our study suggests that lipid oxidation is an important mechanism for PCa progression and may serve as a biological predictor of PCa progression. Introduction: Oxidative stress has been found to be associated with the progression of prostate cancer (PCa); however, human studies which identify differential roles of each oxidation pathway in PCa progression are lacking. We aimed to identify which oxidative stress markers, specifically lipid and global oxidation and glycation, are associated with PCa progression. Patients and Methods: We recruited 3 groups of patients from a urologic clinic at the University of Cincinnati Medical Center: men with PCa who had undergone prostatectomy, men with PCa under watchful waiting, and men with benign prostatic hyperplasia (BPH). We used the most commonly used lipid oxidation marker, F2-isoprostanes; global oxidation markers, fluorescent oxidation products (FlOPs); and the commonly used marker for advanced glycation end products, carboxymethyllysine. These biomarkers were measured in plasma samples at baseline entry. Plasma prostate-specific antigen (PSA) was measured at enrollment and follow-up visits. Results: Compared with men with BPH, men with PCa who had undergone prostatectomy had 26% (P ¼ .01) higher levels of F2-isoprostanes and 20% (P ¼ .08) higher levels of carboxymethyllysine. All the oxidation markers were similar when comparing men under watchful waiting with men with BPH. When examining the associations between baseline oxidation markers and follow-up PSAs, we found that different oxidation markers had differential patterns associated with PSA elevation. F2-isoprostanes were positively associated with PSA elevation among men with PCa; FlOP_320 was positively associated with PSA elevation among both men with PCa and men with BPH, whereas among men with PCa under watchful waiting, FlOP_360 and FlOP_400 had opposite trends of associations with PSA elevation. Conclusions: Our study suggested that high levels of lipid oxidation were associated with PCa progression, whereas different global oxidation markers had different patterns associated with PCa progression. Large-scale clinical studies are needed to confirm our associations. Our study provides a comprehensive view of the relationship between biomarkers and PCa progression. Clinical Genitourinary Cancer, Vol. -, No. -, --- ª 2019 Elsevier Inc. All rights reserved. Keywords: Glycation, Lipid oxidation, Metal oxidation, Oxidative stress, PSA
Introduction Prostate cancer (PCa) is the most common cancer among men, consisting of about 10% of all new cancer cases in the year 2019.1 Although there are many theories regarding the cause of the disease, 1 Division of Epidemiology and Biostatistics, School of Public Health, San Diego State University, San Diego, CA 2 Moores Cancer Center, University of California at San Diego, San Diego, CA 3 Department of Environmental Health, University of Cincinnati Medical School, Cincinnati, OH 4 Department of Surgery, University of Cincinnati Medical School, Cincinnati, OH
Submitted: Jun 22, 2019; Revised: Aug 17, 2019; Accepted: Sep 10, 2019 Address for correspondence: Tianying Wu, MD, PhD, Associate Professor, Division of Epidemiology and Biostatistics, School of Public Health, San Diego State University, Hardy Tower, Rm 172, 5500 Campanile Dr, San Diego, CA 92182-4162 E-mail contact: [email protected]
1558-7673/$ - see frontmatter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.clgc.2019.09.014
oxidative stress is one factor that has been found to be associated with the progression of PCa in animal and cell culture models.2 Oxidative stress causes cell mutations and tissue damage, thereby increasing risk of cancer development.2,3 Biomarkers that can reflect biological mechanism and predict PCa progression are lacking. Moreover, few studies have addressed the role of oxidative stress, specifically lipid and global oxidation and glycation, in the progression of PCa.2,4-6 Previous studies have compared levels of oxidation markers among men with PCa, men with benign prostatic hyperplasia (BPH) and healthy controls; however, these studies have not compared lipid oxidation versus glycation or global oxidation.4,5 Furthermore, these studies did not examine whether these oxidation markers were associated with prostate-specific antigen (PSA) levels over time, whether these biomarkers had different patterns of associations with PSA levels, and whether these patterns were consistent across men with PCa who
Clinical Genitourinary Cancer Month 2019
-1
Oxidative Biomarkers in Prostate Cancer Progression
2
-
underwent prostatectomy, men with PCa under watchful waiting, or among men with BPH. In one study by Custovic et al,6 researchers measured a lipid oxidation marker, acrolein, in prostate tissue and found that tissue levels of acrolein predicted future recurrence among men with PCa who underwent prostatectomy; however, they measured this marker in prostate tissue but not blood or urine. Circulating biospecimens such as blood or urine are easier to obtain than tissue, as the majority of the tissues collected from surgery are needed for pathology reports. As a result, there is great need to identify specific biomarkers that reflect the biological mechanism to predict PCa progression among men who have had prostatectomy who are still at risk of PCa recurrence. Approximately 30% to 50% of men with PCa after prostatectomy will have elevated PSA levels within 10 years.7 Although PSA levels should be undetectable following prostatectomy, elevated PSA levels are associated with PCa progression, recurrence, and/or metastatic disease.7 Until recently, the underlying mechanism for PSA elevations is not clear. If we identify an oxidation marker that can predict PSA elevation, it will help us identify a potential mechanism and create strategies for preventing PCa progression and recurrence among men who have had a prostatectomy. To better assess oxidative stress in humans, we selected a comprehensive panel of biomarkers to measure oxidation products, because oxidation products can be generated from multiple pathways including lipid, protein, DNA, and carbohydrate oxidation. Currently, the measurement of F2-isoprostanes is the most commonly used marker for lipid oxidation and has been found to be higher in patients with PCa than in control groups.8 Additionally, advanced glycation end products (AGEs) are generated in the presence of high levels of glucose and from glucose oxidation. AGEs accumulate in tissues over time as part of the normal aging process and glucose oxidation can promote cancer development.2,9,10 Moreover, we are also interested in global levels of oxidation that encompass oxidation products from multiple pathways. Our lab has previously identified a biomarker, namely, fluorescent oxidation products (FlOPs), which reflects oxidation from multiple pathways including lipid, protein, DNA, and carbohydrate oxidation.11 This marker has been found to be 10 times more sensitive than the thiobarbituric acid reactive substances (TBARS) assay, which is a commonly used oxidation assay for measuring malondialdehyde.12 Because men who have had prostatectomy are still at risk for PCa progression and recurrence, it would be worthwhile to determine which oxidation pathway influences cancer stages and cancer progression. For instance, we previously found that high levels of AGEs future PCa events, whereas F2-isoprostanes were not associated with future PCa events among healthy men.2 F2isoprostanes have been found to be higher in men who already have PCa but not before they have a PCa diagnosis (pre-cancerous stage), compared with healthy controls.8 There have been limited studies that examine which oxidation pathways play a role in PCa progression among men with prostatectomies and whether they are associated with stages of PCa and urologic conditions. The objectives of this research were to: (1) examine whether AGEs, F2isoprostanes, and FlOPs were associated with PSA levels among men with PCa who have undergone a prostatectomy, among men with PCa under watchful waiting, and among men with BPH; and (2) compare levels of these oxidation markers across these 3 groups.
Clinical Genitourinary Cancer Month 2019
Patients and Methods Patient Recruitment and Sample Collection After approval was obtained from the Institutional Review Board at the University of Cincinnati Medical Center, written informed consent was collected from patients from the urologic clinic at the University of Cincinnati Medical Center. A total of 43 men between 50 and 85 years of age were recruited from 2010 to 2011, with a follow-up time of 4 years. These men were divided into 3 groups: men with PCa who had undergone a prostatectomy; men with PCa who were under watchful waiting; and men with BPH only. Inclusion criteria were: men aged 50 to 85 years with pathologically confirmed PCa or BPH who had a PSA test during diagnosis, at the time of blood draw, and planned to have follow-up PSA testing. Patients with PCa were either considered under watchful waiting or had prostatectomy only and not had any new surgery or treatment. Exclusion criteria were: men younger than 50 years or older than 85 years, had additional cancer diagnoses other than PCa, and unable to provide inform consent. Patients with PCa who had chemotherapy or radiation therapy were also excluded. Among these 43 men, 46.5% (n ¼ 20) of patients had BPH, 37% (n ¼ 16) were patients with PCa who had a prostatectomy, and 18.6% (n ¼ 7) were patients with PCa under watchful waiting. Among the 43 men, 16% (n ¼ 7) were Black, and the remaining 84% (n ¼ 36) were White. Data on PCa diagnosis, prostatectomy status, BPH condition, and comorbidities such as diabetes and hypertension were collected through medical record review. Gleason scores were also obtained through pathologic reports. All 43 patients had PSA testing at the urologic clinic at baseline enrollment. The PSA test was performed to monitor any progression or recurrence among patients with PCa, BPH progression, or to detect possible PCa among men with BPH. PSA levels were also tested at follow-up clinical visits among study participants. Blood samples for measurement of biomarkers of interest (AGEs, F2-isoprostanes, and FlOPs) were collected at baseline enrollment. Necessary precautions were taken to ensure data validity and prevent influences of measurements of oxidation biomarkers from blood samples. To avoid data extraction and reporting bias, medical and demographic data were extracted by a medical coordinator, and data were entered by research students who were blinded to the purpose of this study. For blood samples, the main concern was whether the delay in processing blood samples would influence the measurements of oxidation biomarkers. For instance, levels of F-2-isoprostanes would increase if the blood samples were not processed within 24 hours after blood collection, even if they were stored on 4 C ice packs before processing.13 Measurement of FlOPs13,14 and AGEs (unpublished data) did not change even after 24 hours after blood collection, if samples were stored in a 4 C refrigerator or on ice packs before processing. Our collected blood samples were stored in a refrigerator at the clinics immediately after collection and these samples were transferred on ice to Dr Wu’s lab for processing. The isolated plasma samples were stored in a 80 C freezer within 3 hours after blood collection. Our blood collection procedures assured that measurements of these biomarkers were not influenced by blood collection methods.
Measurement of Oxidation Biomarkers We measured plasma levels of lipid oxidation using a commonly used marker, F2-isoprostanes; a novel global oxidation identified in our lab, FlOPs; and AGEs, using the commonly used marker of AGEs, carboxymethyllysine (CML). These biomarkers were
Tianying Wu et al Table 1 Baseline Characteristics Among Men With PCa Who Have Had Prostatectomy, Men With PCa Under Watchful Waiting, and Men With BPH Variables
PCa With Prostatectomy (n [ 16), %
PCa Under Watchful Waiting (n [ 7), %
BPH (n [ 20), %
69.3 (9.6)
71.6 (6.3)
67.0 (5.9) 100
Mean age, y (SD) Race White
75
86
Black
25
14
Mean body mass index, kg/m2 (SD)
28.9 (5.1)
0
29.34 (1.5)
27.0 (3.8) 40
History of hypertension
69
71
History of diabetes
19
43
20
Transurethral resection of prostate
12.5
0
20
Family history of PCa Smoking
25
29
15
Never smoked
69
29
75
Past smoker
19
29
20
Current smoker
13
43
PSA levels at initial blood draw, ng/mLa
5
0.1 (0.1, 1.5)
5.8 (0.6, 8.0)
3.6 (2.3, 4.6)
0.1 (0.1, 1.0)
6.0 (0.6, 8.4)
3.2 (1.8, 4.3)
Time difference between the first follow-up PSA and PSA at blood draw, y
0.5 (0.4, 0.9)
0.5 (0.5, 0.7)
0.5 (0.2, 0.6)
Multiple PSA levels, ng/mLc
0.1 (0.1, 1.5)
6.7 (4.8, 7.8)
4.6 (3.4, 7.5)
3.8 (2.1, 10.6)b
NA
3.4 (0.4-6.4)b
First follow-up PSA levels, ng/mLa a
Time between surgery and blood collection dates, ya
Abbreviations: BPH ¼ benign prostatic hyperplasia; NA ¼ not available; PCa ¼ prostate cancer; PSA ¼ prostate-specific antigen; SD ¼ standard deviation. a Values are medians (quartile 1 and quartile 3). b The time between surgery and blood collection was counted from the date of prostatectomy to blood collection for men who underwent prostatectomy and was counted from the date of transurethral resection of prostate to blood draw; only 4 men had transurethral resection of prostate among the BPH group. c Multiple PSA levels include PSA measures at initial blood draw, first follow-up, and any PSA measures after first follow-up if available.
measured at baseline when patients were enrolled. The measurement of these biomarkers has been described in detail in our previous publications.2 Briefly, F2-isoprostanes was measured by gas chromatography/negative ion chemical ionization mass spectrometry, and measurement of FlOPs was extracted using plasma with ethanol/ether (3:1, v/v). Fluorescence was determined with a fluorescent spectrophotometer. The excitation/emission wavelengths were 360/420 nm for FlOP_360, 320/420 nm for FlOP_320, and 400/475 nm for FlOP_400. FlOP_360 represents oxidation products that are generated from oxidized phospholipids or from lipid oxidation products reacting with proteins, DNA, and carbohydrates in presence of phospholipids.12 FlOP_320 is formed when oxidation products such as lipid hydroperoxides, aldehydes, and ketones react with DNA in the presence of metals.12 Finally, FlOP_400 reflects the interaction between malondialdehyde (MDA), proteins, and phospholipid.12 Plasma CML was measured using an enzymelinked immunosorbent assay using monoclonal anti-CML antibody (6D12) and a secondary antibody, alkaline phosphate labeled antimouse lgG1.2
Statistical Analyses One-way analysis of variance with Bonferroni-multiple comparison adjustment were used to compare the levels of oxidation markers between men with PCa after prostatectomy, men with PCa under watchful waiting, and men with BPH. Furthermore, we compared men with PCa with Gleason scores < 7 and 7 to further assess the association of the aggressiveness of PCa with oxidation levels. Linear mixed model was also used to analyze the association between
biomarkers and PSA levels over time. The relationships of logtransformed PSA with oxidation biomarkers and time were analyzed with the mixed effect regression models, in which the logtransformed PSA was the dependent variable and biomarkers of oxidative stress, time, and interaction term between biomarkers of oxidative stress and time were independent variables. Multiple PSA measurements (2-6 measurements) at different time points were included in this analysis. Beta coefficients and P values were estimated in the models. Additionally, we adjusted for medications used for hypertension and diabetes and time between surgery and blood draw to determine if the estimates changed or remained constant. All analyses were conducted in SAS 9.3 (SAS Institute, Cary, NC).
Results Baseline Characteristics As shown in Table 1, the average age in these 3 groups of men was between 67 and 72 years. Additionally, all study participants were overweight, with a body mass index over 25 kg/m2, ranging from 27 to 29 kg/m2 among all 3 groups. In each group, at least 40% of the population had hypertension, and at least 20% had diabetes. These comorbidities, along with history of smoking, are potentially associated with oxidative stress. There was also a family history of PCa (15%-29%) among these groups. The time between blood collection and surgery for patients with PCa who underwent prostatectomy and men with BPH were 3.8 years and 3.4 years, respectively. The time was determined from the date of prostatectomy to blood draw for men who underwent prostatectomy, whereas the time was determined from transurethral resection of prostate to blood draw for men
Clinical Genitourinary Cancer Month 2019
-3
Oxidative Biomarkers in Prostate Cancer Progression Table 2 Baseline Levels of Oxidation Markers Across Men With PCa Who Have Had Prostatectomy, Men With PCa Under Watchful Waiting, and Men Who Have BPH PCa With Prostatectomy (n [ 16)
BPH (n [ 20) Value (Reference)
Variables AGEs, ug/mLa F2 isoprostane, ng/mLa F1OP_360, fluorescent intensity/mLb F1OP_320, fluorescent intensity/mL F1OP_400 (fluorescent intensity/ml)
260 0.046 32 145 9
(91) (0.012) (23, 41) (59, 246) (7, 11)
P Value Compared With BPH
Value 313 0.058 30 121 9
PCa Under Watchful Waiting (n [ 7)
(82) (0.015) (25, 40) (77, 216) (7, 11)
.080 .012 .508 1.000 1.000
Value 309 0.048 30 74 7
P Value Compared With BPH
(31) (0.011) (27, 35) (73, 934) (7, 8)
.187 .743 .749 .749 .041
Bold value is statistically significant (P < .05). Abbreviations: AGEs ¼ advanced glycation end products; BPH ¼ benign prostatic hyperplasia; FlOP ¼ fluorescent oxidation products; PCa ¼ prostate cancer. a Biomarkers that were normally distributed are presented as means (standard deviation). b Biomarkers that were not normally distributed are presented as medians (quartile 1, quartile 3).
with BPH. The median PSA levels at initial blood draw (ng/mL) among men with PCa after prostatectomy, under watchful waiting, and with BPH were 0.1, 5.8, and 3.6, respectively. Based on summary data of multiple follow-up PSA levels, the median PSA levels increased over time for men with BPH and men with PCa under watchful waiting but not for men who underwent prostatectomy. Although the median PSA levels did not change over time, PSA levels did increase over time in some men who underwent prostatectomy (data not shown). Furthermore, approximately 78% of men with PCa who underwent prostatectomy had at least 1 times of PSA > 0.2 ng/mL during follow-up visits (data not shown). Additionally, among men with PCa who had prostatectomy and men with BPH, 12.5% and 20% had transurethral resection of prostate, respectively.
Comparisons of Levels of Oxidation Markers Across the 3 Groups In Table 2, we found that AGEs levels were 20% higher (P ¼ .08) and F-2 isoprostanes were 26% higher (P ¼ .012) in men with PCa who have had prostatectomy when compared with men with BPH. However, we did not find any significant differences on FlOP markers between these 2 groups. Lastly, men with PCa who are under watchful waiting had 22% lower levels of FlOP_400 (P ¼ .041) than men with BPH.
Comparisons of Levels of Oxidation Markers Among Men With PCa Who Had Prostatectomy In Table 3, among men with PCa who had prostatectomy, we found that AGEs, as measured by CML, were 25% lower among
those who had a Gleason score 7 (P ¼ .026) than those who had a Gleason score < 7.
Baseline Oxidation Markers With Follow-up PSA Level We further analyzed the associations between these biomarkers of interest and follow-up PSA levels among men who had at least 2 PSA blood draws (PSA at initial blood draw and first follow-up PSA after blood draw). To further show results of these associations, 3 models were created. Model 1 included only biomarkers, time, and the interaction term between time and biomarkers. Model 2 further adjusted for medications and time between surgery and blood draw. Model 3 and model 2 included similar variables, with the exception that time between surgery and blood draw were removed because very few men with BPH had surgery, and none of the patients under watchful waiting had surgery. We found that baseline F2isoprostanes were positively associated with follow-up PSA levels in men with PCa who had prostatectomy and among men with PCa under watchful waiting in model 1, model 2, and model 3. The beta-estimate for the F2-isoprostatens (dependent variable) and longitudinal follow-up PSA (multiple PSAs) was 95.39 (P < .0006) for men with PCa who had prostatectomy (in model 2) and 26.57 (P ¼ .002) for men with PCa under watchful waiting (in model 3). FLOP_320 was positively associated with longitudinal follow-up PSA levels in all 3 groups in the final model (model 2 or model 3): beta-estimate ¼ 0.20 (P ¼ .06) for BPH, beta-estimate ¼ 0.85 (P ¼ .08) for men with PCa who underwent prostatectomy, and beta-estimate ¼ 0.35 (P ¼ .003) for men with PCa under watchful waiting. FlOP_360 was inversely associated with longitudinal
Table 3 The Levels of Oxidative Markers Among Participants With Prostate Cancer With Prostatectomy Stratified by Gleason Score (<7 Compared With ‡ 7) Gleason Score < 7 (n [ 5)
Variables a
AGEs, ug/mL F2 isoprostane, ng/mLa F1OP_360, fluorescent intensity/mL F1OP_320, fluorescent intensity/mL F1OP_400, fluorescent intensity/mL
378 0.052 25.95 78.42 8.38
Gleason Score ‡ 7 (n [ 11)
(111) (0.009) (22.22, 31.24) (60.04, 93.13) (6.44, 9.54)
Bold value is statistically significant (P < .05). Abbreviations: AGEs ¼ advanced glycation end products; FlOP ¼ fluorescent oxidation products. a Values are means (standard deviation). Other variables are presented as medians (quartile 1, quartile 3).
4
-
Clinical Genitourinary Cancer Month 2019
284 0.060 31.70 143.84 9.77
(45) (0.017) (26.28, 41.12) (96.91, 233.96) (7.35, 11.12)
P Value .026 .315 .602 .117 .602
Tianying Wu et al Table 4 Baseline Oxidation Markers Associated With Prostate-specific Antigen Levels Over Time With Oxidation Biomarkers
Variables AGEs F2-isoprostane FlOP_360 FlOP_320 FlOP_400
BPH (n [ 12)
Prostatectomy (n [ 13)
Watchful Waiting (n [ 4)
Beta (P Value)
Beta (P Value)
Beta (P Value)
Model 1 0.00 9.15 0.15 0.04 0.42
(.92) (.31) (.53) (.73) (.15)
Model 3 0.00 3.74 0.21 0.20 0.09
(.07) (.60) (.33) (.06) (.71)
Model 1 0.01 103.17 1.34 0.11 L3.29
(.31) (<.001) (.34) (.85) (.007)
Model 2 0.01 95.39 1.02 0.46 1.50
(.41) (.0006) (.40) (.08) (.36)
Model 1 0.007 26.93 0.33 0.02 3.29
(.45) (.002) (.78) (.85) (.07)
Model 3 L0.04 26.57 L2.62 0.35 11.40
(.0009) (.002) (.005) (.003) (.0002)
In model 1, biomarkers (AGEs, F2-isoprostanes, FlOP_360, FlOP_320 or FlOP_400), time and the interaction between time and biomarkers were adjusted. All these biomarkers were not adjusted simultaneously but separately in the model. In model 2, covariates included variables in model 1, plus medications used for hypertension and diabetes and time between surgery and blood draw. In model 3, covariates included the variables in model 1 plus medications used for hypertension and diabetes. Time between surgery and blood draw was not included in model 3, because there were no patients who had surgery in men under the watchful waiting group and only a few patients had surgeries in the BPH group. Abbreviations: AGEs ¼ advanced glycation end products; BPH ¼ benign prostatic hyperplasia; FlOP ¼ fluorescent oxidation products.
follow-up PSA in all 3 groups but only statistically significant in men with PCa under watchful waiting (beta-estimate ¼ 2.62; P ¼ .005). FlOP_400 was only significantly and positively associated with longitudinal follow-up PSA in men with PCa under watchful waiting (beta-estimate ¼ 11.40; P ¼ .0002). AGEs levels were inversely and significantly associated with longitudinal followup PSA levels in men with PCa under watchful waiting (betaestimate ¼ 0.04; P ¼ .0009). In addition, we found that F2-isoprostanes levels were significantly associated with baseline PSA levels (beta-estimate ¼ 79; P ¼ .04) in men with PCa who had prostatectomy but not in men with PCa under watchful waiting (data not shown).
Discussion We have found that lipid oxidation marker, F2-isoprostanes, significantly predicted future PSA levels among men who had prostatectomy and that this association was also significant among men under watchful waiting. AGEs (glycation marker) was only significantly and inversely associated with future PSA levels among men with PCa with watchful waiting. Different global oxidation products had different patterns associated with future PSA levels. FlOP_320 was positively associated with future PSA levels in men with PCa and men with BPH, whereas FlOP_360 and FlOP_400 had opposite trends of association with future PSA levels in men with PCa under watchful waiting. AGEs and F2-isoprostanes were higher in men with PCa who had undergone prostatectomy than men with BPH, whereas high levels of AGEs were found in men with PCa with lower Gleason scores. We examined a comprehensive profile of oxidation markers, with each having differential patterns with PCa progression and severity. This study suggests that lipid oxidation is an important mechanism for PCa progression, and high levels of glycation were associated with lower grades of PCa and inversely associated with PCa progression among men under watchful waiting. Our study findings have also been supported by other experimental studies.15e18 Measurement of F2-isoprostanes has been shown to significantly predict future PSA levels among men who had prostatectomy in our study; one explanation for the increase of F2-isoprostanes can be owing to increased de novo lipid synthesis in PCa cells.18 Additionally, androgens, which are risk factors of PCa progression, have also been demonstrated to increase lipogenic enzymes.19 Interestingly, animal studies had
demonstrated that inhibition of lipid oxidation upregulated glucose metabolism, whereas increased lipid oxidation downregulated glucose metabolism.17 Although abnormal glucose metabolism may or may not be associated with increased AGEs, this may partially explain why we found that men who had advanced stages of PCa (higher Gleason scores) had lower glycation (low AGEs levels).18 Our study indicates that lipid oxidation is associated with PCa progression, whereas glycation prohibits PCa progression among men with advanced stages of PCa. Large-scale prospective studies are needed to confirm our findings. The results from FlOP_360, FlOP_320 and FlOP_400 suggest that global oxidation markers may have differential patterns associated with PCa progression. FlOP_320 represents global oxidation products generated in presence of metals.12,15,20 Studies have shown that metals such as iron and copper were higher in malignant tissues than benign prostate tissues.21 It has been hypothesized that metalinitiated carcinogenesis is through the promotion of oxidation and DNA damage.21,22 Our results support this hypothesis. Measurement of FlOP_320 may better reflect oxidative damage caused by metals than measurement of free metals in plasma, because free metals are low as they tend to bind with protein.22 Furthermore, oxidative stress may have a bidirectional relationship with cancer as moderate levels of oxidation can promote tumor growth, whereas very high levels of oxidation can lead to cancer cell death.12,23 Compared with other specific oxidation markers, FlOPs reflect global levels of oxidation. We observed an inverse association between FlOP_360 with PSA levels over time among our 3 groups, although this was only statistically significant in men with PCa under watchful waiting. Furthermore, among men with PCa under watchful waiting, we observed an opposite pattern comparing FlOP_360 with FlOP_400. FlOP_360 and FlOP_400 share similar products but each also has different products; FlOP_360 reflected a combination of oxidation products from lipid, protein, DNA, and carbohydrates, whereas FlOP_400 reflects the interaction between MDA, proteins, and phospholipid.12 MDA are generated mainly from n-3 fatty acids.12 Animal and cell culture studies have shown that n-3 fatty acidsupplementation can reduce PCa growth; however, the results were not consistent in human studies.24-26 These inconsistent results in human studies may be owing to the population investigated, stage of PCa examined, and other environmental factors that may interfere with function of n-3 fatty acids. In our study, we found inconsistent
Clinical Genitourinary Cancer Month 2019
-5
Oxidative Biomarkers in Prostate Cancer Progression trends of FlOP_400 between men with PCa under watchful waiting and men with PCa who had undergone prostatectomy. The latter group may have more advanced stages than the patients who were under watchful waiting. As a result, the stage of PCa may also influence these associations. Nevertheless, understanding the complex patterns of oxidation markers on PCa progression and PCa stage will help us predict future disease progression and create specific therapeutic targets for patients with PCa. Additionally, we found some significant changes of estimates of the associations of oxidation markers with future PSA levels after adjustment of medications used for hypertension and diabetes. Studies have shown that nonsteroidal anti-inflammatory drugs, statins, and thiazide diuretics can reduce PSA levels.27 Medications used for hypertension and diabetes can include some of these medicines. These medicines can also potentially influence oxidation markers.28,29 Among the 3 groups of men in this study, there were approximately 40% to 70% of men who used hypertension drugs and 20% to 40% who used anti-diabetic drugs; thus adjustment of these drugs did change some of the estimates in Table 4. Our study has several limitations. Because of the small sample size, we cannot adjust for enough confounding factors that may alter the oxidation markers and PSA levels. Adjustment of medications and time between surgery and date of blood collection changed some of the estimates; however, confounding by other factors cannot be ruled out.
Conclusion Although the small sample size is a limitation and presents difficulties in applying these findings to the general population, these results nevertheless help identify which biomarkers are of interest in the progression of PCa. Additionally, the collection of multiple biomarkers of oxidative stress and PSA levels allows for a comprehensive view of the relationship between biomarkers and disease progression. As a result, this study provides a starting point in examining various oxidation biomarkers and their association with PSA levels among men with PCa who have undergone prostatectomy, men with PCa under watchful waiting, and men with BPH. Further studies with a larger sample size are needed to confirm these associations.
Clinical Practice Points The cornerstone of current approaches in monitoring progres-
sion and recurrence after treatment of PCa is measurement of PSA levels. As PSA levels do not uncover biological mechanisms of PCa, targeted therapy cannot be created based on PSA results. This study demonstrated that plasma biomarkers of lipid oxidation were significant predictors of PSA elevation among men with PCa who had undergone prostatectomy. Our study will aid in creating prevention strategies for PCa progression and recurrence. If our results are confirmed in other studies, medications can be designed to inhibit lipid oxidation based on this important evidence.
Acknowledgments This study was supported by National Cancer Institute (KO7 CA138714) and San Diego State University start-up funds.
6
-
Clinical Genitourinary Cancer Month 2019
Disclosure The authors have stated that they have no conflicts of interest.
References 1. National Institues of Health; National Cancer Institute; Surveillence, Epidemiology, and End Results Program. Cancer stat facts: prostate cancer, Available at: https://seer.cancer.gov/statfacts/html/prost.html. Accessed: May 20, 2019. 2. Yang S, Pinney SM, Mallick P, Ho SM, Bracken B, Wu T. Impact of oxidative stress biomarkers and carboxymethyllysine (an advanced glycation end product) on prostate cancer: a prospective study. Clin Genitourin Cancer 2015; 13: 347-51. 3. Luo H, Chiang HH, Louw M, Susanto A, Chen D. Nutrient sensing and the oxidative stress response. Trends Endocrinol Metab 2017; 28:449-60. 4. Miyake H, Hara I, Kamidono S, Eto H. Oxidative DNA damage in patients with prostate cancer and its response to treatment. J Urol 2004; 171:1533-6. 5. Pace G, Di Massimo C, De Amicis D, et al. Oxidative stress in benign prostatic hyperplasia and prostate cancer. Urol Int 2010; 85:328-33. 6. Custovic Z, Zarkovic K, Cindric M, et al. Lipid peroxidation product acrolein as a predictive biomarker of prostate carcinoma relapse after radical surgery. Free Radic Res 2010; 44:497-504. 7. Pound CR, Brawer MK, Partin AW. Evaluation and treatment of men with biochemical prostate-specific antigen recurrence following definitive therapy for clinically localized prostate cancer. Rev Urol 2001; 3:72-84. 8. Barocas DA, Motley S, Cookson MS, et al. Oxidative stress measured by urine F2-isoprostane level is associated with prostate cancer. J Urol 2011; 185:2102-7. 9. Foster D, Spruill L, Walter KR, et al. AGE metabolites: a biomarker linked to cancer disparity? Cancer Epidemiol Biomarkers Prev 2014; 23:2186-91. 10. Turner DP. Advanced glycation end-products: a biological consequence of lifestyle contributing to cancer disparity. Cancer Res 2015; 75:1925-9. 11. Wu T, Willett WC, Rifai N, Rimm EB. Plasma fluorescent oxidation products as potential markers of oxidative stress for epidemiologic studies. Am J Epidemiol 2007; 166:552-60. 12. Frankel EN. Lipid oxidation. Dundee, Scotland. The Oily Press LTD; 2005. 13. Wu T, Rifai N, Roberts LJ 2nd, Willett WC, Rimm EB. Stability of measurements of biomarkers of oxidative stress in blood over 36 hours. Cancer Epidemiol Biomarkers Prev 2004; 13:1399-402. 14. Jensen MK, Wang Y, Rimm EB, Townsend MK, Willett W, Wu T. Fluorescent oxidation products and risk of coronary heart disease: a prospective study in women. J Am Heart Assoc 2013; 2:e000195. 15. Wu X, Daniels G, Lee P, Monaco ME. Lipid metabolism in prostate cancer. Am J Clin Exp Urol 2014; 2:111-20. 16. Schlaepfer IR, Glodé LM, Hitz CA, et al. Inhibition of lipid oxidation increases glucose metabolism and enhances 2-Deoxy-2-[(18)F]Fluoro-D-Glucose uptake in prostate cancer mouse xenografts. Mol Imaging Biol 2015; 17:529-38. 17. Suburu JI, Chen YQ. Lipids and prostate cancer. Prostaglandins Other Lipid Mediat 2012; 98:1-10. 18. Semba RD, Beck J, Sun K, et al. Relationship of a dominant advanced glycation end product, serum carboxymethyl-lysine, and abnormal glucose metabolism in adults: the Baltimore Longitudinal Study of Aging. J Nutr Health Aging 2010; 14: 507-13. 19. Swinnen JV, Esquenet M, Goossens K, Heyns W, Verhoeven G. Androgens stimulate fatty acid synthesis in the human prostate cancer cell line. Cancer Res 1997; 57:1086-90. 20. Yang S, Feskanich D, Willett WC, Eliassen AH, Wu T. Association between global biomarkers of oxidative stress and hip fracture in postmenopausal women: a prospective study. J Bone Miner Res 2014; 29:2577-83. 21. Yaman M, Atici D, Bakirdere S, Akdeniz I. Comparison of trace metal concentrations in malign and benign human prostate. J Med Chem 2005; 48:630-4. 22. Mason M, Yamashita T, Wesson L, Eisenmant G, Eisenberg D. Where metal ions bind in proteins. Proc Nadl Acad Sci 1990; 87:5648-52. 23. Reczek CR, Chandel NS. The two faces of reactive oxygen species in cancer. Ann Rev Cancer Biol 2017; 1:79-98. 24. Li J, Gu Z, Pan Y, et al. Dietary supplementation of a-linolenic acid induced conversion of n-3 LCPUFAs and reduced prostate cancer growth in a mouse model. Lipids Health Dis 2017; 16:136. 25. Cavazos DA, Price RS, Apte SS, deGraffenried LA. Docosahexaenoic acid selectively induces human prostate cancer cell sensitivity to oxidative stress through modulation of NF-kB. Prostate 2011; 71:1420-8. 26. Aucoin M, Cooley K, Knee C, et al. Fish-derived omega-3 fatty acids and prostate cancer: a systematic review. Integr Cancer Ther 2017; 16:32-62. 27. Chang SL, Harshman LC, Presti JC Jr. Impact of common medications on serum total prostate-specific antigen levels: analysis of the National Health and Nutrition Examination Survey. J Clin Oncol 2010; 28:3951-7. 28. Chandran G, Sirajudeen KN, Yusoff NS, Swamy M, Samarendra MS. Effect of the antihypertensive drug enalapril on oxidative stress markers and antioxidant enzymes in kidney of spontaneously hypertensive rat. Oxid Med Cell Longev 2014; 2014:608512. 29. Alrefai AA, Alsalamony AM, Fatani SH, Kamel HFM. Effect of variable antidiabetic treatments strategy on oxidative stress markers in obese patients with T2DM. Diabetol Metab Syndr 2017; 9:27.
Identification of Differential Patterns of Oxidative Biomarkers in Prostate Cancer Progression Tianying Wu,1,2 Susan Kasper,3 Ronnie Meiyi Wong,1 Bruce Bracken4 Abstract This article focuses on prostate cancer (PCa) progression. The study population consisted of patients recruited from urologic clinics at the University of Cincinnati Medical Center. Our study suggests that lipid oxidation is an important mechanism for PCa progression and may serve as a biological predictor of PCa progression. Introduction: Oxidative stress has been found to be associated with the progression of prostate cancer (PCa); however, human studies which identify differential roles of each oxidation pathway in PCa progression are lacking. We aimed to identify which oxidative stress markers, specifically lipid and global oxidation and glycation, are associated with PCa progression. Patients and Methods: We recruited 3 groups of patients from a urologic clinic at the University of Cincinnati Medical Center: men with PCa who had undergone prostatectomy, men with PCa under watchful waiting, and men with benign prostatic hyperplasia (BPH). We used the most commonly used lipid oxidation marker, F2-isoprostanes; global oxidation markers, fluorescent oxidation products (FlOPs); and the commonly used marker for advanced glycation end products, carboxymethyllysine. These biomarkers were measured in plasma samples at baseline entry. Plasma prostate-specific antigen (PSA) was measured at enrollment and follow-up visits. Results: Compared with men with BPH, men with PCa who had undergone prostatectomy had 26% (P ¼ .01) higher levels of F2-isoprostanes and 20% (P ¼ .08) higher levels of carboxymethyllysine. All the oxidation markers were similar when comparing men under watchful waiting with men with BPH. When examining the associations between baseline oxidation markers and follow-up PSAs, we found that different oxidation markers had differential patterns associated with PSA elevation. F2-isoprostanes were positively associated with PSA elevation among men with PCa; FlOP_320 was positively associated with PSA elevation among both men with PCa and men with BPH, whereas among men with PCa under watchful waiting, FlOP_360 and FlOP_400 had opposite trends of associations with PSA elevation. Conclusions: Our study suggested that high levels of lipid oxidation were associated with PCa progression, whereas different global oxidation markers had different patterns associated with PCa progression. Large-scale clinical studies are needed to confirm our associations. Our study provides a comprehensive view of the relationship between biomarkers and PCa progression. Clinical Genitourinary Cancer, Vol. -, No. -, --- ª 2019 Elsevier Inc. All rights reserved. Keywords: Glycation, Lipid oxidation, Metal oxidation, Oxidative stress, PSA
Introduction Prostate cancer (PCa) is the most common cancer among men, consisting of about 10% of all new cancer cases in the year 2019.1 Although there are many theories regarding the cause of the disease, 1 Division of Epidemiology and Biostatistics, School of Public Health, San Diego State University, San Diego, CA 2 Moores Cancer Center, University of California at San Diego, San Diego, CA 3 Department of Environmental Health, University of Cincinnati Medical School, Cincinnati, OH 4 Department of Surgery, University of Cincinnati Medical School, Cincinnati, OH
Submitted: Jun 22, 2019; Revised: Aug 17, 2019; Accepted: Sep 10, 2019 Address for correspondence: Tianying Wu, MD, PhD, Associate Professor, Division of Epidemiology and Biostatistics, School of Public Health, San Diego State University, Hardy Tower, Rm 172, 5500 Campanile Dr, San Diego, CA 92182-4162 E-mail contact: [email protected]
1558-7673/$ - see frontmatter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.clgc.2019.09.014
oxidative stress is one factor that has been found to be associated with the progression of PCa in animal and cell culture models.2 Oxidative stress causes cell mutations and tissue damage, thereby increasing risk of cancer development.2,3 Biomarkers that can reflect biological mechanism and predict PCa progression are lacking. Moreover, few studies have addressed the role of oxidative stress, specifically lipid and global oxidation and glycation, in the progression of PCa.2,4-6 Previous studies have compared levels of oxidation markers among men with PCa, men with benign prostatic hyperplasia (BPH) and healthy controls; however, these studies have not compared lipid oxidation versus glycation or global oxidation.4,5 Furthermore, these studies did not examine whether these oxidation markers were associated with prostate-specific antigen (PSA) levels over time, whether these biomarkers had different patterns of associations with PSA levels, and whether these patterns were consistent across men with PCa who
Clinical Genitourinary Cancer Month 2019
-1
Oxidative Biomarkers in Prostate Cancer Progression
2
-
underwent prostatectomy, men with PCa under watchful waiting, or among men with BPH. In one study by Custovic et al,6 researchers measured a lipid oxidation marker, acrolein, in prostate tissue and found that tissue levels of acrolein predicted future recurrence among men with PCa who underwent prostatectomy; however, they measured this marker in prostate tissue but not blood or urine. Circulating biospecimens such as blood or urine are easier to obtain than tissue, as the majority of the tissues collected from surgery are needed for pathology reports. As a result, there is great need to identify specific biomarkers that reflect the biological mechanism to predict PCa progression among men who have had prostatectomy who are still at risk of PCa recurrence. Approximately 30% to 50% of men with PCa after prostatectomy will have elevated PSA levels within 10 years.7 Although PSA levels should be undetectable following prostatectomy, elevated PSA levels are associated with PCa progression, recurrence, and/or metastatic disease.7 Until recently, the underlying mechanism for PSA elevations is not clear. If we identify an oxidation marker that can predict PSA elevation, it will help us identify a potential mechanism and create strategies for preventing PCa progression and recurrence among men who have had a prostatectomy. To better assess oxidative stress in humans, we selected a comprehensive panel of biomarkers to measure oxidation products, because oxidation products can be generated from multiple pathways including lipid, protein, DNA, and carbohydrate oxidation. Currently, the measurement of F2-isoprostanes is the most commonly used marker for lipid oxidation and has been found to be higher in patients with PCa than in control groups.8 Additionally, advanced glycation end products (AGEs) are generated in the presence of high levels of glucose and from glucose oxidation. AGEs accumulate in tissues over time as part of the normal aging process and glucose oxidation can promote cancer development.2,9,10 Moreover, we are also interested in global levels of oxidation that encompass oxidation products from multiple pathways. Our lab has previously identified a biomarker, namely, fluorescent oxidation products (FlOPs), which reflects oxidation from multiple pathways including lipid, protein, DNA, and carbohydrate oxidation.11 This marker has been found to be 10 times more sensitive than the thiobarbituric acid reactive substances (TBARS) assay, which is a commonly used oxidation assay for measuring malondialdehyde.12 Because men who have had prostatectomy are still at risk for PCa progression and recurrence, it would be worthwhile to determine which oxidation pathway influences cancer stages and cancer progression. For instance, we previously found that high levels of AGEs future PCa events, whereas F2-isoprostanes were not associated with future PCa events among healthy men.2 F2isoprostanes have been found to be higher in men who already have PCa but not before they have a PCa diagnosis (pre-cancerous stage), compared with healthy controls.8 There have been limited studies that examine which oxidation pathways play a role in PCa progression among men with prostatectomies and whether they are associated with stages of PCa and urologic conditions. The objectives of this research were to: (1) examine whether AGEs, F2isoprostanes, and FlOPs were associated with PSA levels among men with PCa who have undergone a prostatectomy, among men with PCa under watchful waiting, and among men with BPH; and (2) compare levels of these oxidation markers across these 3 groups.
Clinical Genitourinary Cancer Month 2019
Patients and Methods Patient Recruitment and Sample Collection After approval was obtained from the Institutional Review Board at the University of Cincinnati Medical Center, written informed consent was collected from patients from the urologic clinic at the University of Cincinnati Medical Center. A total of 43 men between 50 and 85 years of age were recruited from 2010 to 2011, with a follow-up time of 4 years. These men were divided into 3 groups: men with PCa who had undergone a prostatectomy; men with PCa who were under watchful waiting; and men with BPH only. Inclusion criteria were: men aged 50 to 85 years with pathologically confirmed PCa or BPH who had a PSA test during diagnosis, at the time of blood draw, and planned to have follow-up PSA testing. Patients with PCa were either considered under watchful waiting or had prostatectomy only and not had any new surgery or treatment. Exclusion criteria were: men younger than 50 years or older than 85 years, had additional cancer diagnoses other than PCa, and unable to provide inform consent. Patients with PCa who had chemotherapy or radiation therapy were also excluded. Among these 43 men, 46.5% (n ¼ 20) of patients had BPH, 37% (n ¼ 16) were patients with PCa who had a prostatectomy, and 18.6% (n ¼ 7) were patients with PCa under watchful waiting. Among the 43 men, 16% (n ¼ 7) were Black, and the remaining 84% (n ¼ 36) were White. Data on PCa diagnosis, prostatectomy status, BPH condition, and comorbidities such as diabetes and hypertension were collected through medical record review. Gleason scores were also obtained through pathologic reports. All 43 patients had PSA testing at the urologic clinic at baseline enrollment. The PSA test was performed to monitor any progression or recurrence among patients with PCa, BPH progression, or to detect possible PCa among men with BPH. PSA levels were also tested at follow-up clinical visits among study participants. Blood samples for measurement of biomarkers of interest (AGEs, F2-isoprostanes, and FlOPs) were collected at baseline enrollment. Necessary precautions were taken to ensure data validity and prevent influences of measurements of oxidation biomarkers from blood samples. To avoid data extraction and reporting bias, medical and demographic data were extracted by a medical coordinator, and data were entered by research students who were blinded to the purpose of this study. For blood samples, the main concern was whether the delay in processing blood samples would influence the measurements of oxidation biomarkers. For instance, levels of F-2-isoprostanes would increase if the blood samples were not processed within 24 hours after blood collection, even if they were stored on 4 C ice packs before processing.13 Measurement of FlOPs13,14 and AGEs (unpublished data) did not change even after 24 hours after blood collection, if samples were stored in a 4 C refrigerator or on ice packs before processing. Our collected blood samples were stored in a refrigerator at the clinics immediately after collection and these samples were transferred on ice to Dr Wu’s lab for processing. The isolated plasma samples were stored in a 80 C freezer within 3 hours after blood collection. Our blood collection procedures assured that measurements of these biomarkers were not influenced by blood collection methods.
Measurement of Oxidation Biomarkers We measured plasma levels of lipid oxidation using a commonly used marker, F2-isoprostanes; a novel global oxidation identified in our lab, FlOPs; and AGEs, using the commonly used marker of AGEs, carboxymethyllysine (CML). These biomarkers were
Tianying Wu et al Table 1 Baseline Characteristics Among Men With PCa Who Have Had Prostatectomy, Men With PCa Under Watchful Waiting, and Men With BPH Variables
PCa With Prostatectomy (n [ 16), %
PCa Under Watchful Waiting (n [ 7), %
BPH (n [ 20), %
69.3 (9.6)
71.6 (6.3)
67.0 (5.9) 100
Mean age, y (SD) Race White
75
86
Black
25
14
Mean body mass index, kg/m2 (SD)
28.9 (5.1)
0
29.34 (1.5)
27.0 (3.8) 40
History of hypertension
69
71
History of diabetes
19
43
20
Transurethral resection of prostate
12.5
0
20
Family history of PCa Smoking
25
29
15
Never smoked
69
29
75
Past smoker
19
29
20
Current smoker
13
43
PSA levels at initial blood draw, ng/mLa
5
0.1 (0.1, 1.5)
5.8 (0.6, 8.0)
3.6 (2.3, 4.6)
0.1 (0.1, 1.0)
6.0 (0.6, 8.4)
3.2 (1.8, 4.3)
Time difference between the first follow-up PSA and PSA at blood draw, y
0.5 (0.4, 0.9)
0.5 (0.5, 0.7)
0.5 (0.2, 0.6)
Multiple PSA levels, ng/mLc
0.1 (0.1, 1.5)
6.7 (4.8, 7.8)
4.6 (3.4, 7.5)
3.8 (2.1, 10.6)b
NA
3.4 (0.4-6.4)b
First follow-up PSA levels, ng/mLa a
Time between surgery and blood collection dates, ya
Abbreviations: BPH ¼ benign prostatic hyperplasia; NA ¼ not available; PCa ¼ prostate cancer; PSA ¼ prostate-specific antigen; SD ¼ standard deviation. a Values are medians (quartile 1 and quartile 3). b The time between surgery and blood collection was counted from the date of prostatectomy to blood collection for men who underwent prostatectomy and was counted from the date of transurethral resection of prostate to blood draw; only 4 men had transurethral resection of prostate among the BPH group. c Multiple PSA levels include PSA measures at initial blood draw, first follow-up, and any PSA measures after first follow-up if available.
measured at baseline when patients were enrolled. The measurement of these biomarkers has been described in detail in our previous publications.2 Briefly, F2-isoprostanes was measured by gas chromatography/negative ion chemical ionization mass spectrometry, and measurement of FlOPs was extracted using plasma with ethanol/ether (3:1, v/v). Fluorescence was determined with a fluorescent spectrophotometer. The excitation/emission wavelengths were 360/420 nm for FlOP_360, 320/420 nm for FlOP_320, and 400/475 nm for FlOP_400. FlOP_360 represents oxidation products that are generated from oxidized phospholipids or from lipid oxidation products reacting with proteins, DNA, and carbohydrates in presence of phospholipids.12 FlOP_320 is formed when oxidation products such as lipid hydroperoxides, aldehydes, and ketones react with DNA in the presence of metals.12 Finally, FlOP_400 reflects the interaction between malondialdehyde (MDA), proteins, and phospholipid.12 Plasma CML was measured using an enzymelinked immunosorbent assay using monoclonal anti-CML antibody (6D12) and a secondary antibody, alkaline phosphate labeled antimouse lgG1.2
Statistical Analyses One-way analysis of variance with Bonferroni-multiple comparison adjustment were used to compare the levels of oxidation markers between men with PCa after prostatectomy, men with PCa under watchful waiting, and men with BPH. Furthermore, we compared men with PCa with Gleason scores < 7 and 7 to further assess the association of the aggressiveness of PCa with oxidation levels. Linear mixed model was also used to analyze the association between
biomarkers and PSA levels over time. The relationships of logtransformed PSA with oxidation biomarkers and time were analyzed with the mixed effect regression models, in which the logtransformed PSA was the dependent variable and biomarkers of oxidative stress, time, and interaction term between biomarkers of oxidative stress and time were independent variables. Multiple PSA measurements (2-6 measurements) at different time points were included in this analysis. Beta coefficients and P values were estimated in the models. Additionally, we adjusted for medications used for hypertension and diabetes and time between surgery and blood draw to determine if the estimates changed or remained constant. All analyses were conducted in SAS 9.3 (SAS Institute, Cary, NC).
Results Baseline Characteristics As shown in Table 1, the average age in these 3 groups of men was between 67 and 72 years. Additionally, all study participants were overweight, with a body mass index over 25 kg/m2, ranging from 27 to 29 kg/m2 among all 3 groups. In each group, at least 40% of the population had hypertension, and at least 20% had diabetes. These comorbidities, along with history of smoking, are potentially associated with oxidative stress. There was also a family history of PCa (15%-29%) among these groups. The time between blood collection and surgery for patients with PCa who underwent prostatectomy and men with BPH were 3.8 years and 3.4 years, respectively. The time was determined from the date of prostatectomy to blood draw for men who underwent prostatectomy, whereas the time was determined from transurethral resection of prostate to blood draw for men
Clinical Genitourinary Cancer Month 2019
-3
Oxidative Biomarkers in Prostate Cancer Progression Table 2 Baseline Levels of Oxidation Markers Across Men With PCa Who Have Had Prostatectomy, Men With PCa Under Watchful Waiting, and Men Who Have BPH PCa With Prostatectomy (n [ 16)
BPH (n [ 20) Value (Reference)
Variables AGEs, ug/mLa F2 isoprostane, ng/mLa F1OP_360, fluorescent intensity/mLb F1OP_320, fluorescent intensity/mL F1OP_400 (fluorescent intensity/ml)
260 0.046 32 145 9
(91) (0.012) (23, 41) (59, 246) (7, 11)
P Value Compared With BPH
Value 313 0.058 30 121 9
PCa Under Watchful Waiting (n [ 7)
(82) (0.015) (25, 40) (77, 216) (7, 11)
.080 .012 .508 1.000 1.000
Value 309 0.048 30 74 7
P Value Compared With BPH
(31) (0.011) (27, 35) (73, 934) (7, 8)
.187 .743 .749 .749 .041
Bold value is statistically significant (P < .05). Abbreviations: AGEs ¼ advanced glycation end products; BPH ¼ benign prostatic hyperplasia; FlOP ¼ fluorescent oxidation products; PCa ¼ prostate cancer. a Biomarkers that were normally distributed are presented as means (standard deviation). b Biomarkers that were not normally distributed are presented as medians (quartile 1, quartile 3).
with BPH. The median PSA levels at initial blood draw (ng/mL) among men with PCa after prostatectomy, under watchful waiting, and with BPH were 0.1, 5.8, and 3.6, respectively. Based on summary data of multiple follow-up PSA levels, the median PSA levels increased over time for men with BPH and men with PCa under watchful waiting but not for men who underwent prostatectomy. Although the median PSA levels did not change over time, PSA levels did increase over time in some men who underwent prostatectomy (data not shown). Furthermore, approximately 78% of men with PCa who underwent prostatectomy had at least 1 times of PSA > 0.2 ng/mL during follow-up visits (data not shown). Additionally, among men with PCa who had prostatectomy and men with BPH, 12.5% and 20% had transurethral resection of prostate, respectively.
Comparisons of Levels of Oxidation Markers Across the 3 Groups In Table 2, we found that AGEs levels were 20% higher (P ¼ .08) and F-2 isoprostanes were 26% higher (P ¼ .012) in men with PCa who have had prostatectomy when compared with men with BPH. However, we did not find any significant differences on FlOP markers between these 2 groups. Lastly, men with PCa who are under watchful waiting had 22% lower levels of FlOP_400 (P ¼ .041) than men with BPH.
Comparisons of Levels of Oxidation Markers Among Men With PCa Who Had Prostatectomy In Table 3, among men with PCa who had prostatectomy, we found that AGEs, as measured by CML, were 25% lower among
those who had a Gleason score 7 (P ¼ .026) than those who had a Gleason score < 7.
Baseline Oxidation Markers With Follow-up PSA Level We further analyzed the associations between these biomarkers of interest and follow-up PSA levels among men who had at least 2 PSA blood draws (PSA at initial blood draw and first follow-up PSA after blood draw). To further show results of these associations, 3 models were created. Model 1 included only biomarkers, time, and the interaction term between time and biomarkers. Model 2 further adjusted for medications and time between surgery and blood draw. Model 3 and model 2 included similar variables, with the exception that time between surgery and blood draw were removed because very few men with BPH had surgery, and none of the patients under watchful waiting had surgery. We found that baseline F2isoprostanes were positively associated with follow-up PSA levels in men with PCa who had prostatectomy and among men with PCa under watchful waiting in model 1, model 2, and model 3. The beta-estimate for the F2-isoprostatens (dependent variable) and longitudinal follow-up PSA (multiple PSAs) was 95.39 (P < .0006) for men with PCa who had prostatectomy (in model 2) and 26.57 (P ¼ .002) for men with PCa under watchful waiting (in model 3). FLOP_320 was positively associated with longitudinal follow-up PSA levels in all 3 groups in the final model (model 2 or model 3): beta-estimate ¼ 0.20 (P ¼ .06) for BPH, beta-estimate ¼ 0.85 (P ¼ .08) for men with PCa who underwent prostatectomy, and beta-estimate ¼ 0.35 (P ¼ .003) for men with PCa under watchful waiting. FlOP_360 was inversely associated with longitudinal
Table 3 The Levels of Oxidative Markers Among Participants With Prostate Cancer With Prostatectomy Stratified by Gleason Score (<7 Compared With ‡ 7) Gleason Score < 7 (n [ 5)
Variables a
AGEs, ug/mL F2 isoprostane, ng/mLa F1OP_360, fluorescent intensity/mL F1OP_320, fluorescent intensity/mL F1OP_400, fluorescent intensity/mL
378 0.052 25.95 78.42 8.38
Gleason Score ‡ 7 (n [ 11)
(111) (0.009) (22.22, 31.24) (60.04, 93.13) (6.44, 9.54)
Bold value is statistically significant (P < .05). Abbreviations: AGEs ¼ advanced glycation end products; FlOP ¼ fluorescent oxidation products. a Values are means (standard deviation). Other variables are presented as medians (quartile 1, quartile 3).
4
-
Clinical Genitourinary Cancer Month 2019
284 0.060 31.70 143.84 9.77
(45) (0.017) (26.28, 41.12) (96.91, 233.96) (7.35, 11.12)
P Value .026 .315 .602 .117 .602
Tianying Wu et al Table 4 Baseline Oxidation Markers Associated With Prostate-specific Antigen Levels Over Time With Oxidation Biomarkers
Variables AGEs F2-isoprostane FlOP_360 FlOP_320 FlOP_400
BPH (n [ 12)
Prostatectomy (n [ 13)
Watchful Waiting (n [ 4)
Beta (P Value)
Beta (P Value)
Beta (P Value)
Model 1 0.00 9.15 0.15 0.04 0.42
(.92) (.31) (.53) (.73) (.15)
Model 3 0.00 3.74 0.21 0.20 0.09
(.07) (.60) (.33) (.06) (.71)
Model 1 0.01 103.17 1.34 0.11 L3.29
(.31) (<.001) (.34) (.85) (.007)
Model 2 0.01 95.39 1.02 0.46 1.50
(.41) (.0006) (.40) (.08) (.36)
Model 1 0.007 26.93 0.33 0.02 3.29
(.45) (.002) (.78) (.85) (.07)
Model 3 L0.04 26.57 L2.62 0.35 11.40
(.0009) (.002) (.005) (.003) (.0002)
In model 1, biomarkers (AGEs, F2-isoprostanes, FlOP_360, FlOP_320 or FlOP_400), time and the interaction between time and biomarkers were adjusted. All these biomarkers were not adjusted simultaneously but separately in the model. In model 2, covariates included variables in model 1, plus medications used for hypertension and diabetes and time between surgery and blood draw. In model 3, covariates included the variables in model 1 plus medications used for hypertension and diabetes. Time between surgery and blood draw was not included in model 3, because there were no patients who had surgery in men under the watchful waiting group and only a few patients had surgeries in the BPH group. Abbreviations: AGEs ¼ advanced glycation end products; BPH ¼ benign prostatic hyperplasia; FlOP ¼ fluorescent oxidation products.
follow-up PSA in all 3 groups but only statistically significant in men with PCa under watchful waiting (beta-estimate ¼ 2.62; P ¼ .005). FlOP_400 was only significantly and positively associated with longitudinal follow-up PSA in men with PCa under watchful waiting (beta-estimate ¼ 11.40; P ¼ .0002). AGEs levels were inversely and significantly associated with longitudinal followup PSA levels in men with PCa under watchful waiting (betaestimate ¼ 0.04; P ¼ .0009). In addition, we found that F2-isoprostanes levels were significantly associated with baseline PSA levels (beta-estimate ¼ 79; P ¼ .04) in men with PCa who had prostatectomy but not in men with PCa under watchful waiting (data not shown).
Discussion We have found that lipid oxidation marker, F2-isoprostanes, significantly predicted future PSA levels among men who had prostatectomy and that this association was also significant among men under watchful waiting. AGEs (glycation marker) was only significantly and inversely associated with future PSA levels among men with PCa with watchful waiting. Different global oxidation products had different patterns associated with future PSA levels. FlOP_320 was positively associated with future PSA levels in men with PCa and men with BPH, whereas FlOP_360 and FlOP_400 had opposite trends of association with future PSA levels in men with PCa under watchful waiting. AGEs and F2-isoprostanes were higher in men with PCa who had undergone prostatectomy than men with BPH, whereas high levels of AGEs were found in men with PCa with lower Gleason scores. We examined a comprehensive profile of oxidation markers, with each having differential patterns with PCa progression and severity. This study suggests that lipid oxidation is an important mechanism for PCa progression, and high levels of glycation were associated with lower grades of PCa and inversely associated with PCa progression among men under watchful waiting. Our study findings have also been supported by other experimental studies.15e18 Measurement of F2-isoprostanes has been shown to significantly predict future PSA levels among men who had prostatectomy in our study; one explanation for the increase of F2-isoprostanes can be owing to increased de novo lipid synthesis in PCa cells.18 Additionally, androgens, which are risk factors of PCa progression, have also been demonstrated to increase lipogenic enzymes.19 Interestingly, animal studies had
demonstrated that inhibition of lipid oxidation upregulated glucose metabolism, whereas increased lipid oxidation downregulated glucose metabolism.17 Although abnormal glucose metabolism may or may not be associated with increased AGEs, this may partially explain why we found that men who had advanced stages of PCa (higher Gleason scores) had lower glycation (low AGEs levels).18 Our study indicates that lipid oxidation is associated with PCa progression, whereas glycation prohibits PCa progression among men with advanced stages of PCa. Large-scale prospective studies are needed to confirm our findings. The results from FlOP_360, FlOP_320 and FlOP_400 suggest that global oxidation markers may have differential patterns associated with PCa progression. FlOP_320 represents global oxidation products generated in presence of metals.12,15,20 Studies have shown that metals such as iron and copper were higher in malignant tissues than benign prostate tissues.21 It has been hypothesized that metalinitiated carcinogenesis is through the promotion of oxidation and DNA damage.21,22 Our results support this hypothesis. Measurement of FlOP_320 may better reflect oxidative damage caused by metals than measurement of free metals in plasma, because free metals are low as they tend to bind with protein.22 Furthermore, oxidative stress may have a bidirectional relationship with cancer as moderate levels of oxidation can promote tumor growth, whereas very high levels of oxidation can lead to cancer cell death.12,23 Compared with other specific oxidation markers, FlOPs reflect global levels of oxidation. We observed an inverse association between FlOP_360 with PSA levels over time among our 3 groups, although this was only statistically significant in men with PCa under watchful waiting. Furthermore, among men with PCa under watchful waiting, we observed an opposite pattern comparing FlOP_360 with FlOP_400. FlOP_360 and FlOP_400 share similar products but each also has different products; FlOP_360 reflected a combination of oxidation products from lipid, protein, DNA, and carbohydrates, whereas FlOP_400 reflects the interaction between MDA, proteins, and phospholipid.12 MDA are generated mainly from n-3 fatty acids.12 Animal and cell culture studies have shown that n-3 fatty acidsupplementation can reduce PCa growth; however, the results were not consistent in human studies.24-26 These inconsistent results in human studies may be owing to the population investigated, stage of PCa examined, and other environmental factors that may interfere with function of n-3 fatty acids. In our study, we found inconsistent
Clinical Genitourinary Cancer Month 2019
-5
Oxidative Biomarkers in Prostate Cancer Progression trends of FlOP_400 between men with PCa under watchful waiting and men with PCa who had undergone prostatectomy. The latter group may have more advanced stages than the patients who were under watchful waiting. As a result, the stage of PCa may also influence these associations. Nevertheless, understanding the complex patterns of oxidation markers on PCa progression and PCa stage will help us predict future disease progression and create specific therapeutic targets for patients with PCa. Additionally, we found some significant changes of estimates of the associations of oxidation markers with future PSA levels after adjustment of medications used for hypertension and diabetes. Studies have shown that nonsteroidal anti-inflammatory drugs, statins, and thiazide diuretics can reduce PSA levels.27 Medications used for hypertension and diabetes can include some of these medicines. These medicines can also potentially influence oxidation markers.28,29 Among the 3 groups of men in this study, there were approximately 40% to 70% of men who used hypertension drugs and 20% to 40% who used anti-diabetic drugs; thus adjustment of these drugs did change some of the estimates in Table 4. Our study has several limitations. Because of the small sample size, we cannot adjust for enough confounding factors that may alter the oxidation markers and PSA levels. Adjustment of medications and time between surgery and date of blood collection changed some of the estimates; however, confounding by other factors cannot be ruled out.
Conclusion Although the small sample size is a limitation and presents difficulties in applying these findings to the general population, these results nevertheless help identify which biomarkers are of interest in the progression of PCa. Additionally, the collection of multiple biomarkers of oxidative stress and PSA levels allows for a comprehensive view of the relationship between biomarkers and disease progression. As a result, this study provides a starting point in examining various oxidation biomarkers and their association with PSA levels among men with PCa who have undergone prostatectomy, men with PCa under watchful waiting, and men with BPH. Further studies with a larger sample size are needed to confirm these associations.
Clinical Practice Points The cornerstone of current approaches in monitoring progres-
sion and recurrence after treatment of PCa is measurement of PSA levels. As PSA levels do not uncover biological mechanisms of PCa, targeted therapy cannot be created based on PSA results. This study demonstrated that plasma biomarkers of lipid oxidation were significant predictors of PSA elevation among men with PCa who had undergone prostatectomy. Our study will aid in creating prevention strategies for PCa progression and recurrence. If our results are confirmed in other studies, medications can be designed to inhibit lipid oxidation based on this important evidence.
Acknowledgments This study was supported by National Cancer Institute (KO7 CA138714) and San Diego State University start-up funds.
6
-
Clinical Genitourinary Cancer Month 2019
Disclosure The authors have stated that they have no conflicts of interest.
References 1. National Institues of Health; National Cancer Institute; Surveillence, Epidemiology, and End Results Program. Cancer stat facts: prostate cancer, Available at: https://seer.cancer.gov/statfacts/html/prost.html. Accessed: May 20, 2019. 2. Yang S, Pinney SM, Mallick P, Ho SM, Bracken B, Wu T. Impact of oxidative stress biomarkers and carboxymethyllysine (an advanced glycation end product) on prostate cancer: a prospective study. Clin Genitourin Cancer 2015; 13: 347-51. 3. Luo H, Chiang HH, Louw M, Susanto A, Chen D. Nutrient sensing and the oxidative stress response. Trends Endocrinol Metab 2017; 28:449-60. 4. Miyake H, Hara I, Kamidono S, Eto H. Oxidative DNA damage in patients with prostate cancer and its response to treatment. J Urol 2004; 171:1533-6. 5. Pace G, Di Massimo C, De Amicis D, et al. Oxidative stress in benign prostatic hyperplasia and prostate cancer. Urol Int 2010; 85:328-33. 6. Custovic Z, Zarkovic K, Cindric M, et al. Lipid peroxidation product acrolein as a predictive biomarker of prostate carcinoma relapse after radical surgery. Free Radic Res 2010; 44:497-504. 7. Pound CR, Brawer MK, Partin AW. Evaluation and treatment of men with biochemical prostate-specific antigen recurrence following definitive therapy for clinically localized prostate cancer. Rev Urol 2001; 3:72-84. 8. Barocas DA, Motley S, Cookson MS, et al. Oxidative stress measured by urine F2-isoprostane level is associated with prostate cancer. J Urol 2011; 185:2102-7. 9. Foster D, Spruill L, Walter KR, et al. AGE metabolites: a biomarker linked to cancer disparity? Cancer Epidemiol Biomarkers Prev 2014; 23:2186-91. 10. Turner DP. Advanced glycation end-products: a biological consequence of lifestyle contributing to cancer disparity. Cancer Res 2015; 75:1925-9. 11. Wu T, Willett WC, Rifai N, Rimm EB. Plasma fluorescent oxidation products as potential markers of oxidative stress for epidemiologic studies. Am J Epidemiol 2007; 166:552-60. 12. Frankel EN. Lipid oxidation. Dundee, Scotland. The Oily Press LTD; 2005. 13. Wu T, Rifai N, Roberts LJ 2nd, Willett WC, Rimm EB. Stability of measurements of biomarkers of oxidative stress in blood over 36 hours. Cancer Epidemiol Biomarkers Prev 2004; 13:1399-402. 14. Jensen MK, Wang Y, Rimm EB, Townsend MK, Willett W, Wu T. Fluorescent oxidation products and risk of coronary heart disease: a prospective study in women. J Am Heart Assoc 2013; 2:e000195. 15. Wu X, Daniels G, Lee P, Monaco ME. Lipid metabolism in prostate cancer. Am J Clin Exp Urol 2014; 2:111-20. 16. Schlaepfer IR, Glodé LM, Hitz CA, et al. Inhibition of lipid oxidation increases glucose metabolism and enhances 2-Deoxy-2-[(18)F]Fluoro-D-Glucose uptake in prostate cancer mouse xenografts. Mol Imaging Biol 2015; 17:529-38. 17. Suburu JI, Chen YQ. Lipids and prostate cancer. Prostaglandins Other Lipid Mediat 2012; 98:1-10. 18. Semba RD, Beck J, Sun K, et al. Relationship of a dominant advanced glycation end product, serum carboxymethyl-lysine, and abnormal glucose metabolism in adults: the Baltimore Longitudinal Study of Aging. J Nutr Health Aging 2010; 14: 507-13. 19. Swinnen JV, Esquenet M, Goossens K, Heyns W, Verhoeven G. Androgens stimulate fatty acid synthesis in the human prostate cancer cell line. Cancer Res 1997; 57:1086-90. 20. Yang S, Feskanich D, Willett WC, Eliassen AH, Wu T. Association between global biomarkers of oxidative stress and hip fracture in postmenopausal women: a prospective study. J Bone Miner Res 2014; 29:2577-83. 21. Yaman M, Atici D, Bakirdere S, Akdeniz I. Comparison of trace metal concentrations in malign and benign human prostate. J Med Chem 2005; 48:630-4. 22. Mason M, Yamashita T, Wesson L, Eisenmant G, Eisenberg D. Where metal ions bind in proteins. Proc Nadl Acad Sci 1990; 87:5648-52. 23. Reczek CR, Chandel NS. The two faces of reactive oxygen species in cancer. Ann Rev Cancer Biol 2017; 1:79-98. 24. Li J, Gu Z, Pan Y, et al. Dietary supplementation of a-linolenic acid induced conversion of n-3 LCPUFAs and reduced prostate cancer growth in a mouse model. Lipids Health Dis 2017; 16:136. 25. Cavazos DA, Price RS, Apte SS, deGraffenried LA. Docosahexaenoic acid selectively induces human prostate cancer cell sensitivity to oxidative stress through modulation of NF-kB. Prostate 2011; 71:1420-8. 26. Aucoin M, Cooley K, Knee C, et al. Fish-derived omega-3 fatty acids and prostate cancer: a systematic review. Integr Cancer Ther 2017; 16:32-62. 27. Chang SL, Harshman LC, Presti JC Jr. Impact of common medications on serum total prostate-specific antigen levels: analysis of the National Health and Nutrition Examination Survey. J Clin Oncol 2010; 28:3951-7. 28. Chandran G, Sirajudeen KN, Yusoff NS, Swamy M, Samarendra MS. Effect of the antihypertensive drug enalapril on oxidative stress markers and antioxidant enzymes in kidney of spontaneously hypertensive rat. Oxid Med Cell Longev 2014; 2014:608512. 29. Alrefai AA, Alsalamony AM, Fatani SH, Kamel HFM. Effect of variable antidiabetic treatments strategy on oxidative stress markers in obese patients with T2DM. Diabetol Metab Syndr 2017; 9:27.