Changes in Erythrocyte Transmethylation Ability are Predictive Factors for Tumor Prognosis in Prostate Cancer †

Endogenous transmethylation processes proceed via formaldehyde. Endogenous formaldehyde can be found in special bounded forms in animal tissues. The aim was to find non-invasive methods to help establish an early diagnosis of prostate cancer processes, and to search for a reliable examination repertoire to predict metastasis based on genomic hypomethylation that can be found in tumors. Fifty-eight age-matched patients suffering from prostate cancer and 26 healthy volunteers were enrolled in the study. Biochip technique was applied to quantify serum interleukins and growth factors. OPCL method served to determine mobilized formaldehyde, and fluorometric methods were applied to measure Znand free protoporphyrin concentrations in erythrocytes. Spectrophotometric measurements were carried out to evaluate redox homeostasis in plasma. There were no significant differences in most measured laboratory and immune parameters at different early stages of prostate cancer; at the same time, erythrocyte parameters, especially immobilized formaldehyde determination, were promising. The authors assume that hypomethylation detection in erythrocytes might become a predictive factor in the diagnostic panel in the near future. (doi: 10.5562/cca1748)


INTRODUCTION
A large number of people, mostly around the age of 50, suffer from prostate cancer in the world, and so also in Hungary.In Hungary, 4500 prostate cancer patients and 1400 deaths were registered in 2009.According to a 2008 statistical report, prostate cancer is the second cancer-related cause of death for men in the United States. 1 Prostate cancer is silent and has no early warning symptoms.Extensive use of serum prostate specific antigen (PSA) testing has been increasingly reported at earlier stages, although false negative or positive diagnoses can occur.
Treating prostate cancer is a major challenge today.In case of localized cancer tissue in a patient in good condition with more than 10 years of life expectancy, radical surgery is the treatment of choice.Bones are the predominant sites of distant metastases caused by prostate cancer. 2It is still not possible to determine which patients are at high risk of tumor recurrence after primary local therapy and will not benefit from surgery.No more promising indicators are available to distinguish between surgically curable and oncologically treatable prostate cancers.No optimal factor has so far been found for the prognosis, although several authors maintain that IL-6, IL-10, TNF-alpha and VEGF are increased and IL-2 is decreased in case of the appearance of metastasis in glands and bones.][9] Croat.Chem.Acta 84 (2011) 127.
][12] It was earlier clarified that transmethylation processes occur via formaldehyde, mobilized methyl group and not via methyl cation in life systems. 13n our previous studies, significant changes were observed in erythrocyte function in colectomized patients suffering from colon cancer.In adult colectomized patients after 5-10 years of colectomy without metastasis, erythrocyte mobilized formaldehyde was significantly lower than in controls.At the same time, protoporphyrin concentration was low in patients without metastasis, when the diagnosis before operation was Dukes C. HbA1c level correlated significantly with induced free radical level and decreased the antioxidant status of erythrocytes. 14xplanation of the connection between concentration changes of formaldehyde and protoporphyrin is that arginine (38), methionine (65) and lysine (72) near methionine (80) are methylated and thereby coordinated towards the central iron of heme.Consequently, when the transmethylation activity is normal, the hemoglobin structure is optimal. 15alnikow and Zhitkovich found that genetic and epigenetic mechanisms in metal carcinogenesis and cocarcinogenes depend on nickel, arsenic, and chromium toxicity and that these elements cause hypomethylation. 16In previous examinations, we observed that the element status of erythrocytes in patients with prostate cancer was significantly changed compared to controls in cases of Al (1.90 ± 1.67 vs 0.537 ± 0.260), Ni (0.722 ± 0.565 vs 0.265 ± 0.195) and Pb (0.309 ± 0.301 vs 0.094 ± 0.053).These ion concentrations were significantly high in prostate cancer patients with PSA > 9 (Ref.17).At the same time, the erythrocyte protoporphyrin concentration was very high in prostate cancer similarly to cases of gastrointestinal tumors. 18lthough molecular biological examinations give excellent information about the stages of prostate cancer or metastasis, their clinical applications are very expensive and cannot be widely used.
Since the changes in bounded formaldehyde, Znprotoporphyrin and free protoporphyrin concentrations of erythrocytes may be very important indices in tumor anemia, we would like to know whether the determination of transmethylation ability via formaldehyde dimedone adduct can be a predictive factor for progression of prostate tumor in clinical applications.Immune reactivity was also examined in searching for correlations between immune parameters, PSA values and transmethylation processes.

Patients
The study included a total of 58 patients with prostate cancer, 18 had metastatic prostate cancer and 28 were at an early stage.12 outpatients had negative and 12 had positive histological findings with questionable PSA values and laboratory parameters.Age range was the same in patient groups and 26 healthy volunteers: 69 ± 6 / 68 ± 8 years.

Methods
Blood samples were drawn from the antecubital vein by venipuncture into tubes containing Ca citrate.Plasma, serum and erythrocyte samples were separated by standard methods and stored at −20 °C until assayed.Hemoglobin content was adjusted uniformly to 0.01 g ml −1 for measurements.
Serum routine laboratory parameters were determined with a Hitachi 717 Analyzer, using standard methods (not shown) of Randox.PSA levels were determined using the Abbott MEIA test, and the concentration of CRP was measured with the Olympus immunoturbidimetric test.
Erythrocyte protoporphyrin IX and Zn-protoporphyrin were measured by the fluorometric method using a Jasco FP 6300 type fluorometer (Jasco Co, Japan).Simple micro-method was adapted for purification of protoporphyrin by Chisolm and coworkers (1975). 20xcitation wavelength was 405 nm and that of emission 610 nm.
The reducing power of the sample was determined at 700 nm according to Oyaizu, based on the chemical reaction Fe(III)→Fe(II).Increased absorbance indicated increased reducing power, which was expressed as ascorbic acid equivalent (mmol/l eqAS). 21he hydrogen-donating ability of the sample was estimated in the presence of 1,1-diphenyl-2-picrylhydrazyl radical according to Hatano et al. 22 Free SH-groups were determined by the Sedlack method, based on the Ellmann reaction (1985). 23All spectrophotometic methods were carried out with a Jasco V 550 spectrophotometer (Jasco Co., Japan).
One-way ANOVA statistical analysis was applied to evaluate the significance between patient groups.
This study was approved by the Regional and Institutional Committee of Science and Research Ethics, Semmelweis University, Permission Number TUKEB 127/2006.

RESULTS AND DISCUSSION
Since laboratory diagnosis of prostate cancer is difficult at an early stage and prostate specific antigen can remain in normal range or be falsely positive, the generally used laboratory parameters do not signal the appearance of the disease.IL-1 alpha, -beta, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-alpha, interferon-gamma, vascular endothelial growth factor, monocyte chemoattractant protein-1 and epidermal growth factor concentrations were determined by the biochip technique with an Evidence Biochip Array Analyzer to establish the progression of tumor process.Table 1 does not show any significant differences in most parameters between study groups and controls.Levels of cytokines and growth factors were low at the early stage of prostate cancer.We found concentrations of IL-1 alpha and -beta, IL-10, IFNG (60-80 %) below the measurement range.Serum PSA correlated with serum IL-6 (r = 0.78) but the other markers did not.CRP value was under 5 mg /l in all samples and was not related to the measured parameters.When low concentrations of the above mentioned parameters are found in the sera of patients with prostate cancer, we can suppose an early stage of cancer.Our results show that measurements of cytokines and growth factors did not help establish an early diagnosis of prostate cancer.It is an interesting fact that the concentrations of cytokines and growth factors are significantly lower in old men (age range 60-75 years) than in younger ones (data not published).When we examined the plasma redox parameters of patients suffering from prostate cancer and healthy controls, we observed only moderate alterations.These data did not help establish an early diagnosis and follow cancer progression unambiguously contrasted to inflammatory bowel diseases, where data indicate moderate and serious forms of diseases (Table 2). 24t the same time, erythrocyte parameters, Znprotoporhyrin, free protoporhyrin and bounded HCHO in sensitized dimedone reaction, showed enormous differences compared to healthy controls not only in taxane-treated metastatic but also in histologically negative and positive patients when cancer was first observed.In particular, bounded formaldehyde in erythrocytes showed alterations compared to healthy controls and signaled moderate transmethylation ability at an early stage.Significant differences in this parameter were found between groups.These data showed that transmethylation ability was changed in the course of cancer progression (Table 3).
Early research has already drawn attention to the alterations in DNA methylation, which play an important role in neoplasia. 25DNA hypomethylation leads to elevated mutation rates. 26Nowadays, many review publications reveal that the most studied mechanisms by epigenetics are DNA methylation and histone medications, stable and reversible alterations to the genome that affect gene expression and genome function.The nature and role of the mechanisms of promoter hypermethylation during carcinogenesis have been studied worldwide; however, the mechanism behind one of the earliest epigenetic observations in cancer, genome-wide hypome-thylation, remains unclear. 27Tumor hypermethylation predicts poor prognosis in patients with earlier stages of prostate cancer, and is commonly found in the plasma DNA of patients with castration-resistant prostate cancer. 28,29Extensive molecular biological examinations help us more and more to understand the mechanism of methylation steps, biochemical pathways, key enzyme functions and, recently, to apply targeted therapy. 30,31esearchers are now searching for natural bioactive agents, to modify DNA methylation, such as curcumin, which has a potent DNA hypomethylation property. 32n the basis of our earlier and present studies, hypomethylation seems to be a condition of the system, which can be improved with methyl donating molecules from food ingredients, such as different N-, S-, and O-methylated compounds.The exact mechanisms are not known, but the effectiveness can be checked by laboratory parameters and improved transmethylation ability of erythrocytes in cancer patients. 18,33

CONCLUSION
To conclude, we can say that the determination of transmethylation ability in erythrocytes and plasma PSA levels may constitute a new methodical approach to detecting prostate cancer at an early stage, follow its progression and apply the most suitable therapy.The authors suppose that exhaustion of the system methyl donor pool causes hypomethylation in DNA and histone.This fact is reflected in erythrocyte function in cancer processes.The found connections can be in con-

Table 2 .
Plasma redox parameters of oncological treated cancer patients at different stages

Table 3 .
Erythrocyte parameters of oncological treated cancer patients at different stages b) nv -value is in normal range (normal value of PSA is 0.01-4.00ng/ml).withserioustumor anemia in metastatic carcinoma.Although the preliminary results presented in this paper are very promising, further studies on a larger number of patients have to be carried out to assess the safety and reliability of this method.ABBREVIATIONSCRP, C reactive protein; EGF, epidermal growth factor; HbA1c, glycated hemoglobin HCHO, formaldehyde; ID-1, DNA-binding protein inhibitor ID-1 (inhibitor of differentiation-1); IFNG, interferon-gamma; IL-1 alpha/beta, IL-2, IL-4, IL-6, IL-8, IL-10, interleukins; OPLC, overpressure layer chromatography; PSA, prostate-specific antigen; TNF-alpha, tumor necrosis factor-alpha.