Melatonin receptors, melatonin metabolizing enzymes and cyclin D1 in human breast cancer.

BACKGROUND: Melatonin suppresses breast cancer cell proliferation by inhibiting the upregulation of estrogen-induced cyclin D1 via its G-protein-coupled receptor MT1. Additionally, melatonin stimulates the expression of the estrogen sulfotransferase, SULT1E1. However, metabolism of melatonin via 6-hydroxylation by CYP1A1/1A2 and subsequent sulfonation by SULT1A1/1A3 decreases its intracellular concentration. This could have a negative impact on its oncostatic action in breast cancer. PATIENTS AND METHODS: In this pilot study, we performed immunohistochemical (IHC) analysis of MT1 and cyclin D1 in breast cancer specimens from 33 patients. Also, we investigated the expression of CYP1A1/1A2, SULT1A1/1A3/1E1,and cyclin D1 in cancer (CANC) and adjacent non-cancer (NCANC) specimens from 10 representative breast cancer patients using quantitative real-time reverse transcription polymerase chain reaction. RESULTS: CYP1A1-mRNA-expression was found only in three CANC and in one NCANC. CYP1A2 mRNA was below the detection limit in all patients. SULT1A1 was observed only in two of the 10 CANC and one of the 10 NCANC specimens. But, all 10 CANC and NCANC samples showed high SULT1A3 levels. Cyclin D1 mRNA levels were found in all 10 CANC and NCANC specimens. Furthermore, IHC-staining of cyclin D1 was observed in 27 of 33 CANC and correlated positively with estrogen receptor positivity (p = 0.015). CONCLUSION: The low or even absent expression of CYP1A1 or CYP1A2 in breast cancer specimens suggested that melatonin might be involved in cell cycle arrest.

Quantitative analysis of peroxisome proliferator-activated receptor gamma (PPARgamma) expression in arteries and hearts of patients with ischaemic or dilated cardiomyopathy.

PPARgamma, a nuclear transcription factor, is expressed in various cells within the vasculature and in cardiomyocytes. It has been suggested that PPARgamma is involved in atherogenesis and in cardiac hypertrophy. Therefore, we sought to quantify PPARgamma mRNA in coronary arteries, the aorta and left ventricular specimens from patients with ischaemic (CHD) and dilated cardiomyopathy (CMP). Using real-time PCR, we were able to demonstrate the expression of PPARgamma in all of the human specimens. The lowest expression of PPARgamma was detected in the aorta specimens of both groups (this was set to one). In comparison, the expression in coronary arteries was 2.32-fold in CHD- and 3.78-fold in CMP specimens and in the left ventricle specimens, 2.12-fold in CHD- and 3.51-fold in CMP. Samples from CHD patients showed a higher expression of PPARgamma in all of the samples compared to those from CMP patients (aorta: 1.99-fold; coronary arteries: 1.35; left ventricles: 1.23). PPARgamma levels were not significantly correlated to CD 36 expression values in any group, suggesting that higher levels of PPARgamma are not principally due to increased PPARgamma expression in macrophages. This was confirmed by immunohistochemical analysis, which showed that PPARgamma is also located in the smooth muscle layer and in cardiomyocytes. In conclusion, our observations of increased PPAR mRNA expression in the coronary arteries and left ventricles from CHD and CMP patients suggest an important function of this nuclear receptor in the pathogenesis of heart disease.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in human heart ventricles.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand activated transcription factor which regulates gene expression in various tissues. PPARgamma was primarily found to be associated with lipid and glucose metabolism. Recent experimental studies provided evidence that PPARgamma is also expressed in the arterial wall and in cardiomyocytes and described PPARgamma as a transducer of antihypertropic signaling in the heart. This comparative study sought to investigate whether PPARgamma is differently expressed in the aorta, coronary arteries and left ventricle specimens derived from healthy heart donors (n = 5). By using quantitative PCR, we found that PPARgamma is expressed in all of the human specimens with the by far highest expression (5.01-fold) in the left ventricles compared to aorta, whereas no significant difference was detected between coronary arteries (0.93-fold) vs. aorta. Furthermore, especially great interindividual variations were observed in PPARgamma expression in aorta, and to a lesser extent, in coronary arteries and left ventricle specimens. In conclusion, our data argue for the prominent role of PPARgamma in the human heart, particularly in the normal left ventricle.

Clock gene expression in different synovial cells of patients with rheumatoid arthritis and osteoarthritis.

Patients with rheumatoid arthritis (RA) show modulated circadian rhythms of inflammatory cytokines and cortisol, which may be associated with a modified expression of clock genes. The expression of major clock genes was previously studied in synovial tissues and fibroblasts of patients with RA and osteoarthritis (OA). We therefore especially aimed to examine the localization of clock genes at the cellular level in synovial tissue. Furthermore we were interested in studying the expression of the D site of albumin promoter (albumin D-box) binding protein (DBP) at the immunohistochemical level in human samples. Methods used include the in situ expression of the clock genes Brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal 1), Circadian Locomotor Output Cycles Kaput (Clock), Period 1 and 2 (Per 1 and Per 2), and DBP was examined by immunohistochemistry in synovial tissues of patients with RA or OA. Additionally, expression profiles of different clock genes were determined over 24h by real time PCR in synovial fibroblasts (SFs) after a 2h serum shock or TNF-α. Results show that all clock genes investigated were found to be expressed both in RA and OA synovial tissues. Double staining against cell specific markers revealed that clock proteins were especially seen in macrophages, SFs and B-lymphocytes. Cell counting showed that clock proteins were found in approximately 5-20% of cells. Additionally, preliminary cell culture experiments showed that TNF-α treatment resulted in differential 24h expression profiles between RA and OA samples and also compared to the results obtained from the serum shock experiments. From our study we conclude that the major clock genes, including DBP, are expressed in samples from RA and OA patients, especially in macrophages and synovial fibroblasts, but also in B-lymphocytes. Preliminary experiments suggest that TNF-α seems to be able to modify clock gene expression in synovial fibroblasts.

Short-term effect of macronutrient composition and glycemic index of a yoghurt breakfast on satiety and mood in healthy young men.

BACKGROUND: Promoting satiety and repressing appetite is one major goal in the dietetic therapy of obesity. In the past, several studies investigated the effect of different macronutrients, especially protein and carbohydrates, on short- and long-term satiety in humans. This paper aims to directly compare the effect of protein, rolled oats (low glycemic index), sugar or cornflakes (high glycemic index), and walnuts (high amount of omega-3 fatty acids) as ingredients of a yoghurt breakfast on short-term hunger and satiety in one setting. A second objective was to study the effect of these yoghurt breakfasts on mental state. METHODS: 14 healthy male volunteers participated in this randomized, controlled, cross-over design study. After consuming the different test meals, volunteers repeatedly completed 2 questionnaires over a total of 3 h. RESULTS: The protein meal showed the highest satiety scores and the controls (low-calorie yoghurt) the lowest. The other test meals were not different among each other. Regarding mental state (mood, fatigue, and calmness), no significant difference between the test meals and the low-calorie control was observed. CONCLUSION: The glycemic index does not seem to modify satiety in this short-term setting. The similar mental state between low- and high-calorie breakfasts deserves further investigations.

Time dependence of estrogen receptor expression in human hearts.

OBJECTIVES AND AIMS: Transcriptional effects of estrogens are primarily mediated by the two nuclear estrogen receptors (ER), ERalpha and ERbeta. Both receptors are present in the vasculature and in the human heart and have been shown to act antiatherogenic and to be protective against the development of cardiac hypertrophy. The aim was to quantify ER mRNA expression in left ventricular specimens from patients with coronary heart disease (CHD, n=15) and dilated cardiomyopathy (CMP, n=38) and compare their levels with those from healthy heart donors (n=9). Additionally, a possible variation of ERmRNA expression in human hearts in respect to time of day was studied. METHODS AND RESULTS: mRNA expression of both ER receptors was detected by real-time PCR in all of the human specimens. There was no difference in the relative quantity of the receptors between CHD and CMP patients. However, control specimens showed significant lower levels of either receptor in the healthy myocardium (p<.001 each). Analyzing the time dependency of receptor expression with a cosinor analysis showed a significant 8-hour period rhythm for ERbeta in CMP- but no rhythm in CHD patients. Due to the low patient number, rhythmic analysis was not possible in controls. CONCLUSIONS: The increased ERalpha and ERbeta mRNA expression in left ventricular specimens from CHD and CMP patients might reflect a compensatory mechanism to counteract the decline in ventricular function. Furthermore, we provided evidence for a time dependent variation of ERbeta receptor expression in the human heart.

Clock genes display rhythmic expression in human hearts.

Thus far, clock genes in the heart have been described only in rodents, and alterations of these genes have been associated with various myocardial malfunctions. In this study, we analyzed the expression of clock genes in human hearts. Left papillary muscles of 16 patients with coronary heart disease, 39 subjects with cardiomyopathy, and 9 healthy donors (52 males and 12 females, mean age 55.7+/-11.2; 16-70 yrs) were obtained during orthotopic heart transplantation. We assessed the mRNA levels of PER1, PER2, BMAL1, and CRY1 by real time PCR and analyzed their rhythmic expression by sliding means and Cosinor functions. Furthermore, we sought for differences between the three groups (by ANOVAs) for both the total 24 h period and separate time bins. All four clock genes were expressed in human hearts. The acrophases (circadian rhythm peak time) of the PER mRNAs occurred in the morning (PER1: 07:44 h [peak level 187% higher than trough, p = .008]; PER2: 09:42 h [peak 254% higher than trough, p < .0001], and BMAL1 mRNA in the evening at 21:44 h [peak 438% higher than trough; p < .0001]. No differences were found in the rhythmic patterns between the three groups. No circadian rhythm was detected in CRY1 mRNA in any group. PER1, PER2, and BMAL1 mRNAs revealed clear circadian rhythms in the human heart, with their staging being in antiphase to those in rodents. The circadian amplitudes of the mRNA clock gene levels in heart tissue are more distinct than in any other human tissue so far investigated. The acrophase of the myocardial PER mRNAs and the trough of the myocardial BMAL1 coincide to the time of day of most frequent myocardial incidents.

The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia.

Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT-PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT-PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin-embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5-hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1-mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.

The melatonin receptor subtype MT2 is present in the human cardiovascular system.

We showed that the melatonin receptor subtype, MT1, is expressed in healthy and diseased human coronary arteries. As studies in experimental animals suggest that the MT2 melatonin receptor subtype is also present in the vasculature, we investigated whether the MT2 is expressed in human aorta and coronary arteries. Additionally, MT2 expression in human ventricular specimens was analysed, as melatonin was shown to affect myocyte function. Expression of the MT2-receptor was studied in sections of isolated coronary arteries, aorta and left ventricular specimens from healthy heart donors (control) and patients with dilated or ischemic cardiomyopathy. MT2 expression was found by reverse transcriptase (RT)-nested-polymerase chain reaction (PCR) in all of the specimens (aorta, left ventricle and coronary arteries) derived from controls. Also, visible evidence for receptor expression was found in 12 of 15 samples from cardiomyopathy patients and 10 of 15 of coronary heart disease patients. Additionally, the expression of MT2-receptor between aorta, left ventricle and coronary arteries varied among the individuals, some of them showing highest expression in the aorta while in others principal expression sites were coronary arteries or left ventricles. In conclusion, the MT2-receptor subtype is present in human arteries and left ventricles and it is suggested that in coronary heart disease MT2-receptor expression is altered. Furthermore, there is evidence for heterogeneous MT2 expression patterns in individual patients.