Urinary melatonin-sulfate/cortisol ratio and the presence of prostate cancer: A case-control study

The circadian-related hormones, melatonin and cortisol, have oncostatic and immunosuppressive properties. This study examined the relationship between these two biomarkers and the presence of prostate cancer. We measured their major metabolites in urine collected from 120 newly diagnosed prostate cancer patients and 240 age-matched controls from January 2011 to April 2014. Compared with patients with lower urinary melatonin-sulfate or melatonin-sulfate/cortisol (MT/C) ratio levels, those with above-median levels were significantly less likely to have prostate cancer (adjusted OR (aOR) = 0.59, 95% CI = 0.35–0.99; aOR = 0.46, 95% CI: 0.27–0.77) or advanced stage prostate cancer (aOR = 0.49, 95% CI = 0.26–0.89; aOR = 0.33, 95% CI = 0.17–0.62). The combined effect of both low MT/C ratios and PSA levels exceeding 10 ng/ml was an 8.82-fold greater likelihood of prostate cancer and a 32.06-fold greater likelihood of advanced stage prostate cancer, compared to those with both high MT/C ratios and PSA levels less than 10 ng/ml. In conclusion, patients with high melatonin-sulfate levels or a high MT/C ratio were less likely to have prostate cancer or advanced stage prostate. Besides, a finding of a low MT/C ratio combined with a PSA level exceeding 10 ng/ml showed the greatest potential in detecting prostate cancer and advanced stage prostate cancer.

The fact that patients with prostate cancer have lower melatonin levels than patients with BPH suggests that melatonin may protect against disease severity 23 . Melatonin has been found to have potential in the chemoprevention and treatment of prostate cancer in vitro studies 24 . Cortisol is another hormone important to circadian regulation 25,26 . One previous study revealed that cortisol secretion patterns may be impacted by shift work, which is known to cause circadian disruption 27 . This hormone has been found to influence cancer risk through its effects on immune function 28,29 . It is thus possible that carcinogenicity of circadian disruption can broadly affect many sites among both men and women via melatonin and cortisol levels. Therefore, we conducted this case-control study to examine the relationship between two urine biomarkers of circadian regulation hormones, melatonin and cortisol level, and the presence of prostate cancer and their impacts on the clinical stages of prostate cancer. Because PSA is also associated with the presence or relapse of prostate cancer, this antigen was also included to examine the interaction effect of circadian regulation hormones and PSA on prostate cancer.

Results
In total, we recruited 137 pathology-proved prostate cancer patients and 274 male controls (average: 70.53 years; range 45 to 85 years) between January 2011 and April 2014. After excluding cases with other cancers or cases without available individual matches, we were left with 120 cases and 240 controls to include in our final analysis. As can be seen in Table 1, summary of participant characteristics, the only significant difference between the cases and controls were PSA levels (p < 0.001). Most (75.0%) of those included in the study were 65 years old or older, had high school or college educations or less (76.7%), were married (87.7%), had no family history of prostate cancer (85.5%), did not smoke (70.1%), use alcohol (88.8%), chew betel nut (98.3%), or use vitamin D supplements (96.6%). There were no significant differences between the two groups with regard to these characteristics. Figure 1 shows the differences in the urinary biomarkers between cases and controls. Compared to the controls, the cases had a significantly lower levels of the urinary melatonin-sulfate (mean ± SD) ( After adjusting for other covariates, we found that subjects with high urinary melatonin-sulfate or MT/C ratios were significantly less likely to have prostate cancer compared to those with low urinary melatonin-sulfate or MT/C ratios (adjusted OR (aOR) = 0.59, 95% CI = 0.35-0.99 and aOR = 0.46, 95% CI = 0.27-0.77, respectively) ( addition, subjects with both high MT/C ratio and a pre-operative PSA level exceeding 10 ng/ml were 3.61 fold more likely (95% CI = 1.62-8.07) to have prostate cancer, when compared to those with both high MT/C ratio and a pre-operative PSA level less than 10 ng/ml. ( Table 2). The risk was even higher in the group with low MT/C ratios and pre-operative  Table S1). Categorizing prostate cancer by clinical stage (localized and advanced), we found significant differences of urinary biomarkers when comparing advanced cancer and control groups. No multiplicative scale of interaction was obtained for the MT/C ratio and PSA level (Table 3). In addition, we found evidence of a combined effect of low MT/C ratio and the pre-operative PSA levels exceeding 10 ng/ml in the advanced cancer group compared with either control group or localized cancer group (Table 3). A similar trend pattern was also observed among the subjects when combining melatonin sulfate or cortisol and PSA level (Supplementary Table S2).

Discussion
In this case-control study, we found an inverse relation between first morning urinary melatonin-sulfate levels and the melatonin/cortisol (MT/C) ratio and the presence of prostate cancer overall and advanced stage prostate cancer.
Most prior studies have focused on evaluating the association between melatonin levels and breast cancer. Very few have focused on the association between melatonin and prostate cancer. One cross-sectional study by Bartsch et al. reported that men with prostate cancer had lower melatonin levels than men with BPH 23 . Another case-cohort study in an Icelandic population, conducted by Sigurdardottir et al., found that subjects with below-median morning pre-diagnostic 6-sulfatoxymelatonin (aMT6s) levels had a statistically significant 4-fold increased risk for advanced disease, compared to those with above-median levels (Hazard ratio = 4.04; 95% CI = 1.26-12.98) 30 . However, that study did not find a significant association between morning urinary aMT6s levels and prostate cancer risk overall. Our study contributes additional information about circadian hormones and the presence of prostate cancer in an Asian population.
The protective effect of melatonin on cancer risk may be related to its inhibition of cancer cell growth, its protection of cells from DNA damage, and its promotion of repair of DNA damage once it has occurred 1,31,32 . Recently, Blask et al. conducted a series of experiments using both steroid receptor-positive and -negative human breast cancer xenografts in rats and found an inverse relationship between melatonin level and tumor activity 33,34 . The same research group also reported similar results with prostate cancer xenografts 35 . Other studies have reported a reduction in growth of malignant prostate tumor cells achieved by the administration of both pharmacologic and physiologic doses of melatonin 24,36-38 , although their findings have not always been consistent 39,40 .
Our study also measured cortisol, another important circadian hormone secreted by the adrenal cortex. Cortisol has been found to help regulate both immunity and inflammation; a deficiency in this hormone may result in an unresponsive immune system and an overabundance of the hormone may suppress immune responses 41 . Moreover, chronic dysregulation of the circadian cortisol rhythm has been associated with higher levels of inflammation 28 , and such inflammation may play a critical role in carcinogenesis 42 . Mirick et al. found that circadian disruption can impact cortisol secretion patterns, which may in turn affect cancer risk 27 . Although this study did not find a significant association between one-spot morning urinary cortisol level and prostate cancer, we did find an inverse association between the MT/C ratio and the presence of prostate cancer and advanced stage prostate cancer. Previous studies have found MT/C ratio to be related to different types of depression and the severity of depression 43,44 . This is the first study to evaluate the association between the MT/C ratio and the presence of prostate cancer. It is difficult to decide clinically whether to perform an invasive prostate biopsy for subjects with abnormal PSA levels. Our finding that the combination of low MT/C ratios and PAS levels in excess of 10 ng/ml conferred the highest tendency that a person may have prostate cancer and advanced-stage disease prostate cancer suggests that we might consider the biomarker of MT/C ratio in urine to be an additional tool for judging whether the prostate biopsy is needed or not, when PSA levels are elevated. Further study is needed to investigate the clinical utility of the combining PSA and MT/C to detect the presence of prostate cancer and stage the disease.
A high proportion of controls in this study had PSA levels exceeding10 ng/ml. Increased PSA concentrations are found in the sera of patients with BPH or patients with prostate cancer, respectively 45 . An estimated 50% of men have histologic evidence of BPH by age 50, 75% by age 80, and 90% of men by age 85 years 46 . About 99% of prostate cancer cases occur in those over the age of 50 47 . This is of particular concern in older men, where BPH is more prevalent, as BPH increases gland volume which in turn increases the PSA level as well as the number of sampling errors associated with prostate biopsy 48,49 . For men with PSA 4-10 ng/mL, the detection rate of PCa in Caucasian men may be as high as 40% 50,51 but only 20% in Chinese men 52 . Although the inclusion criteria for our control group was either unremarkable digital rectal exams or remarkable digital rectal exams but histologically confirmed BPH, our control group may have included some men with undiagnosed prostate cancer. However, the inclusion of such patients as controls would only lead to underestimations of the true association.
The strengths of our study are that we were able to consider the most important prostate cancer risk factors, including clinical stage and PSA level in our analyses. Furthermore, we considered two important circadian biomarkers and found the M/C ratio is more relevant to prostate cancer. This study also has several limitations. One limitation is that it is a cross-sectional case-control study, so no clear causal relationship can be inferred. There is a likelihood of a reverse causality between circadian hormones and the presence of prostate cancer, if circadian disruption and/or sleep disruption following a cancer diagnosis results in the decline of melatonin levels. Another limitation is that we used one single measurement of one-spot morning urinary biomarkers, which may not represent long-term exposure level. Although we had broad information on various covariates and were able to control for possible confounders, we still lacked information on factors such as have sleep disorders or medication use, etc. Finally, the exposures of interest were collected using a questionnaire, which may lead to some recall bias.

Conclusion
Lower morning melatonin-sulfate levels or MT/C ratio were associated with the presence of prostate cancer. In addition, patients with both low MT/C ratios and PSA levels exceeding 10 ng/ml appeared to be far more likely to have prostate cancer and advanced stage disease. Because this is a cross-sectional case-control study, we could not establish a definite causal relationship between urinary MT/C ratio and risk of prostate cancer. Larger prospective cohort studies are needed to verify these findings.  Data collection. Overview. Reference date for the cases was defined as date of pathology proof of prostate cancer. Controls were matched within the same month, the reference month. Subjects in both groups answered in-person interviews conducted by trained interviewers using standardized questionnaires collecting socio-demographic information (age, education, and marital status), family history of prostate cancer (PCa) and lifestyle (use of tobacco, alcohol, betel chewing, and vitamin D supplements). The medical charts of all cases were reviewed to collect data on serum prostate-specific antigen (PSA) level at diagnosis and disease stage. The medical charts of controls were reviewed to collect measured PSA results. All study procedures were reviewed and approved by the Institutional Review Board of the Kaohsiung Chung-Ho Memorial Hospital (KMUH-IRB-20110101). All the methods were carried out in accordance with the approved guidelines, and written informed consent was obtained from all subjects.
Urine sample collection. After their interviews, both case and control subjects provided the first-spot morning urine samples after waking up. The samples were divided into labeled cry-tubes of 4.5 mL volume each and then immediately stored at − 20 °C. Urine specimens from case subjects and matched control subjects were handled identically and assayed on the same day and in the same run. All samples were taken out of the freezer simultaneously and sent to the laboratory in the same parcel on dry ice. Laboratory personnel were blinded to the case-control status of all specimens. Analytic error was controlled for by including two standard samples in each assay. The morning urinary measurements showed good sensitivity and specificity in identifying individual differences in nocturnal plasma melatonin levels 53 . Statistical analysis. Urinary melatonin-sulfate/cortisol (MT/C) ratio was calculated by urinary melatonin-sulfate levels divided by urinary cortisol levels as a basis for measuring the combined impact of melatonin and cortisol levels simultaneously on the prostate cancer. Wilcoxon rank-sum test was used to compare the differences of urinary biomarkers, including melatonin-sulfate, cortisol, and MT/C ratio, between case and control groups. In addition, those urinary markers were dichotomized by medians, which were 43.23 ng/mg creatinine for melatonin, 24.06 ng/mg creatinine for cortisol, and 1.76 for MT/C ratio. Unconditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for the association between the urinary markers and the risk of prostate cancer. All analyses were adjusted for age at time of questionnaire completion (< 65 vs. ≥ 65 yrs), family history of PCa, personal habits (smoking, alcohol, and betel nut), and PSA level (< 10 vs. ≥ 10 ng/ml). We also categorized cases into localized prostate cancer (stage T1 or T2) and advanced cancer (extra-prostatic stage T3a or higher, N1/M1) 55 , and used polytomous logistic regression models to determine the risks of developing localized and advanced prostate cancer relative to the control group 56 . Multiplicative interaction was appraised by fitting polytomous logistic models containing categorical variables, as well as their cross-products. Wald Z-tests for cross-product terms were used to evaluate the significance of multiplicative interaction for each pair of comparison. All statistical operations were performed using the SAS 9.1 statistical package; all P-values were two-sided and considered significant if < 0.05.