Stephen Y. W. Shiu

Melatonin-induced inhibition of proliferation and G1/S cell cycle transition delay of human choriocarcinoma JAr cells: Possible involvement of MT2 (MEL1B) receptor

Abstract: Melatonin, the pineal neurohormone, is an evolutionarily conserved photoperiodic signaling molecule with diverse functions that include the entrainment of human circadian rhythms. Although evidence supporting a direct inhibitory action of melatonin on human cancer cell proliferation exists in the literature, the molecular and cellular signaling mechanisms involved are largely undefined. In our study, significant inhibition of human choriocarcinoma JAr cell proliferation at physiological and pharmacological concentrations of melatonin was observed. 2‐Iodomelatonin, a high affinity melatonin receptor agonist, was more potent than melatonin in inhibiting JAr cell proliferation. In addition, the presence of putative melatonin receptors in choriocarcinoma was suggested by the demonstration of specific 2‐[125I]iodomelatonin binding to the tumor. Interestingly, the selective MT2 melatonin receptor ligand, 4‐phenyl‐2‐propionamidotetraline (4‐P‐PDOT), was found to exert not only concentration‐dependent anti‐proliferative actions on JAr cells, but also additive effects with melatonin in inhibiting JAr cell proliferation. Furthermore, MT2 melatonin receptor gene expression by JAr cells was demonstrated by reverse transcription‐polymerase chain reaction (RT‐PCR) and in situ hybridization (ISH). Taken together, our data suggest that the reported anti‐proliferative action of melatonin on human choriocarcinoma JAr cells may be mediated, in part, by MT2 melatonin receptor. Moreover, analysis of melatonin effect on cell cycle kinetics indicated that G1/S transition delay may underlie the observed inhibition of choriocarcinoma cell proliferation by melatonin.

Neuroendocrinology of melatonin in reproduction: recent developments

The circadian melatonin rhythm with high levels in the dark period is important for the synchronization of reproductive response to appropriate environmental conditions in animals. The target sites of melatonin action on reproductive functions remain to be clarified. Using autoradiography (ARG) and radioreceptor binding assays with 2[125I]iodomelatonin, a melatonin agonist, as the radioligand, studies on the sites of melatonin action have increased significantly in the last ten years. The recent cloning of melatonin receptor subtypes also allowed the characterization of receptor(s) to the molecular level. Earlier reports have documented that the hypothalamic-pituitary axis plays a vital role in the regulation of reproduction by melatonin. This is supported in part by the demonstration of melatonin receptors in the suprachiasmatic nuclei (SCN) in the brain and pars tuberalis (PT) in the pituitary. However, the nature of SCN and PT involvement in the reproductive action of melatonin remains unknown. In addition to the hypothalamus and pituitary, the two classical sites of melatonin action, other targets have been identified. The recent demonstration of 2[125I]iodomelatonin binding sites or melatonin receptors in the testis, epididymis, vas deferens, prostate, ovary and mammary gland suggest the concept of multiple sites of melatonin action on the reproductive system. The presence of melatonin receptors in the said tissues is consistent with earlier reports of direct melatonin actions on different levels of the reproductive system. This multiple levels of melatonin action, from the hypothalamus, pituitary, gonads to other reproductive tissues form a robust system of photoperiodic control in animal reproduction. This would guarantee successful gestation and delivery of the offspring at a time with optimum food availability and ultimately favourable for the survival of species. Molecular and cellular studies of melatonin signaling system(s), its regulation and effects on downstream functional events in the future may provide new insights and directions for the study of the physiology and pharmacology of fertility and contraception in animals and humans.

Inhibition of androgen-sensitive LNCaP prostate cancer growth in vivo by melatonin: Association of antiproliferative action of the pineal hormone with mt1 receptor protein expression

Potential involvement of the mt1 receptor in the antiproliferative action of melatonin on androgen‐sensitive LNCaP cells, and melatonin‐induced modulation of androgen‐insensitive PC‐3 cell growth, have been reported in vitro. The effects of melatonin on prostate cancer cell proliferation and their association with mt1 receptor expression were investigated in athymic nude mice xenograft models of LNCaP and PC‐3 cells.

Towards rational and evidence-based use of melatonin in prostate cancer prevention and treatment

Abstract:  Prostate cancer is a public health problem of the elderly men. It has been estimated that one in six men will develop prostate cancer in his lifetime in the USA. There is thus a huge clinical demand for effective therapies for the prevention and treatment of the disease. Here, the scientific evidence supporting the effectiveness of melatonin in inhibiting the development and progression of prostate cancer is reviewed. The rational use of melatonin in prostate cancer prevention, stabilization of clinically localized favourable‐risk prostate cancer and palliative treatment of advanced or metastatic tumour is discussed within the context of the molecular pathogenesis of the disease.

Melatonin slowed the early biochemical progression of hormone-refractory prostate cancer in a patient whose prostate tumor tissue expressed MT1 receptor subtype

Abstract: Melatonin inhibited the proliferation of hormone‐independent LNCaP prostate cancer cells partly via MT1 receptor activation both in vitro and in nude mice xenograft model. In this study, the melatonin receptor expression in the prostate cancer tissue of a patient with bone metastases and the effect of melatonin on the biochemical progression of hormone‐refractory prostate tumor which later developed in the same patient were reported. Saturation and competition 2‐[125I]iodomelatonin binding assays were conducted on prostate tumor tissue obtained by transurethral resection of the prostate from the index patient. The receptor subtype identity of melatonin receptor expressed in the cancer tissue was determined by comparison of the rank order of inhibition constants (Ki) of various melatonergic ligands and the affinity of 4‐phenyl‐2‐propionamidotetraline relative to melatonin in inhibiting 2‐[125I]iodomelatonin binding to the tumor sample and to human cell lines stably transfected with MT1 or MT2 melatonin receptor subtype. MT1 receptor expression in the cancer tissue was also examined by immunohistochemistry. The surgically castrated patient later developed biochemical relapse of his disease. His serum total prostate‐specific antigen (PSA) level was monitored before and during treatment with 5 mg/day oral melatonin at 20:00 hr. High‐affinity (Kd = 103.7 pm) MT1 melatonin receptor subtype was expressed by the patients prostate cancer. As indicated by his PSA levels, melatonin induced stabilization of his hormone‐refractory disease for 6 wk. This report validates melatonins oncostatic action on prostate cancer and the potential involvement of MT1 receptor subtype in the attendant antiproliferative signal transduction as suggested by recent preclinical laboratory findings in a human.

Potential involvement of mt1 receptor and attenuated sex steroid-induced calcium influx in the direct anti-proliferative action of melatonin on androgen-responsive LNCaP human prostate cancer cells

Melatonin, a pineal secretory product, has been shown to exert a direct anti‐proliferative action on the androgen‐sensitive LNCaP prostate cancer cell line through hitherto undefined mechanisms. In this communication, expression of mt1 melatonin receptor protein in human prostate cancer tissues and LNCaP cells was demonstrated by immunohisto(cyto)chemistry and western blotting, hence supporting the use of LNCaP cell line as a model for the study of melatonin signaling in prostate cancer cell growth. Using H‐thymidine incorporation assay, LNCaP cell proliferation was inhibited by 2‐iodomelatonin, a high‐affinity melatonin receptor agonist. Furthermore, melatonin inhibited H‐thymidine incorporation into LNCaP cells and attenuated 5α‐dihydrotestosterone (DHT) or 17β‐estradiol (E2)‐induced stimulation of LNCaP cell proliferation at physiological and pharmacological concentrations. Similar concentration‐dependent inhibition of sex steroid‐induced stimulation of thymidine incorporation into LNCaP cells by 2‐iodomelatonin was also observed. Interestingly, attenuation of sex steroid‐stimulated calcium influx into LNCaP cells by pharmacological concentrations of melatonin was recorded, whereas 2‐iodomelatonin had no effect on cytosolic calcium changes induced by sex steroids. In addition, proliferative and cytosolic calcium changes were associated with inhibition of total prostate‐specific antigen (PSA) production by LNCaP cells at high physiological and pharmacological concentrations of melatonin. Our data suggest that activated mt1 receptor and attenuated sex steroid‐induced calcium influx are two important mechanisms mediating the direct anti‐proliferative action of melatonin on androgen‐responsive human prostate cancer cells.

Signaling mechanisms of melatonin in antiproliferation of hormone-refractory 22Rv1 human prostate cancer cells: Implications for prostate cancer chemoprevention

Abstract:  There is an unmet clinical demand for safe and effective pharmaceuticals/nutraceuticals for prostate cancer prevention and hormone‐refractory prostate cancer treatment. Previous laboratory and human studies of our laboratory demonstrated an association between the antiproliferative action of melatonin and melatonin MT1 receptor expression in prostate cancer. The aim of this study was to determine, using a pharmacological approach, the signaling mechanisms of melatonin in hormone‐refractory 22Rv1 human prostate cancer cell antiproliferation. Both immunoreactive MT1 and MT2 subtypes of G protein‐coupled melatonin receptor were expressed in 22Rv1 cells. Melatonin inhibited, concentration dependently, cell proliferation, upregulated p27Kip1 gene transcription and protein expression, and downregulated activated androgen signaling in 22Rv1 cells. While the effects of melatonin were mimicked by 2‐iodomelatonin, a high‐affinity nonselective MT1 and MT2 receptor agonist, melatonin effects were blocked by luzindole, a nonselective MT1 and MT2 receptor antagonist, but were unaffected by 4‐phenyl‐2‐propionamidotetraline, a selective MT2 receptor antagonist. Importantly, we discovered that the antiproliferative effect of melatonin exerted via MT1 receptor on p27Kip1 gene and protein upregulation is mediated by a novel signaling mechanism involving co‐activation of protein kinase C (PKC) and PKA in parallel. Moreover, we also showed that a melatonin/MT1/PKC mechanism is involved in melatonin‐induced downregulation of activated androgen signal transduction in 22Rv1 cells. Taken together with the known molecular mechanisms of prostate cancer progression and transition to androgen independence, our data provide strong support for melatonin to be a promising small‐molecule useful for prostate cancer primary prevention and secondary prevention of the development and progression of hormone refractoriness.

The effects of melatonin and Ginkgo biloba extract on memory loss and choline acetyltransferase activities in the brain of rats infused intracerebroventricularly with β-amyloid 1–40

Intraventricular infusion of rats with beta-amyloid for 14 days resulted in memory deficit in the water maze as well as decreases in choline acetyltransferase activities and somatostatin levels in the cerebral cortex and hippocampus. These changes were not altered by daily intraperitoneal injection of 20 mg/Kg melatonin. Orally administered Ginkgo biloba extract, however, partially reversed the memory deficit and the decrease in choline actyltransferase activities in the hippocampus. The latter treatment failed to reverse the decrease in somatostatin levels. The results indicate that orally administered Ginkgo biloba extract can protect the brain against beta-amyloid from changes leading to memory deficit through its effect on the cholinergic system.

Biological Basis and Possible Physiological Implications of Melatonin Receptor- Mediated Signaling in the Rat Epididymis

The mammalian epididymis plays an important role in sperm maturation, an important process of male reproduction. Specific high-affinity 2-[125I]iodomelatonin binding sites, satisfying the pharmacokinetic properties of specific receptors, have been found in the rat corpus epididymis, suggesting a direct melatonin action on epididymal physiology. Subsequent molecular and cell biology studies have identified these 2-[125I]iodomelatonin binding sites to be mt1 (MEL1A) and MT2 (MEL1B) melatonin receptor subtypes. Changes in the binding characteristics of these receptors in the rat corpus epididymis in response to castration and steroid hormones like testosterone and hydrocortisone indicated that these membrane melatonin receptors are biologically functional receptors, whose activities are differentially regulated by testosterone and hydrocortisone. These melatonin receptors are coupled to pertussis toxin (PTX)-sensitive Gi protein and probably participate in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions. Furthermore, rat corpus epididymal epithelial cell proliferation was stimulated by melatonin, whose action was dependent on the concentration and duration of exposure to the hormone. Interestingly, an MT2 receptor ligand (4-phenyl-2-propionamidotetraline, 4-P-PDOT) induced a stimulatory effect on epididymal epithelial cell proliferation similar to that produced by melatonin. In contrast, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxo-thiazolidine-2-ylidene]-4-methyl-thiosemi-carbazone, CGP52608) and 8-bromo-cAMP inhibited epididymal epithelial cell proliferation. Taken together, our data lead us to postulate that one of the possible physiological functions of melatonin on the rat epididymis is the stimulation of mt1 and MT2 melatonin receptors resulting in the inhibition of cAMP signaling and an increase in epithelial cell proliferation.

Association between polyomaviruria and microscopic haematuria in bone marrow transplant recipients

The association of polyomaviruria and microscopic haematuria was studied by the use of electron microscopy (EM) and the polymerase chain reaction (PCR) in bone marrow transplant (BMT) recipients. The incidence of BK virus (BKV) and JC virus (JCV) excretion was further elucidated by means of restriction enzyme analysis of the PCR products. Polyomaviruses were detected in 43 (51.2%) of the 84 samples, 13 (30.2%) of which had a virus concentration detectable by EM. By typing with BamHI cleavage, 29 (67.4%) of the 43 positive patients were found to be excreting only BKV and the remaining 14% (32.6%) were excreting both BKV and JCV. Microscopic haematuria was present in 17 (20.2%) of 84 urine samples collected from different patients within 4 months post-transplant. The incidence of microscopic haematuria was significantly higher, 34.9% (P < 0.01), in patients with polyomaviruria than in those without (4.9%) but no difference was observed between the BKV-excreting and BKV/JCV-co-excreting patients. Microscopic haematuria was not present, however, in 53.8 and 65.2% of polyomavirus-excreting patients when virus was detected by EM and PCR respectively. While most episodes of microscopic haematuria observed were self-limiting and asymptomatic, three patients excreting polyomavirus had symptoms of cystitis and one of them had renal impairment that was otherwise unexplained. We thus conclude that polyomaviruses probably contribute to damage of urinary tract tissue in some BMT recipients.