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

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.     

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 3H-thymidine incorporation assay, LNCaP cell proliferation was inhibited by 2-iodomelatonin, a high-affinity melatonin receptor agonist. Furthermore, melatonin inhibited 3H-thymidine incorporation into LNCaP cells and attenuated 5alpha-dihydrotestosterone (DHT) or 17beta-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.     

PKC activation by melatonin modulates vimentin intermediate filament organization in N1E-115 cells

Melatonin enters cells and causes cytoskeletal rearrangements in unicellular organisms, plants and vertebrates. This pineal secretory product causes microtubule enlargement and neurite outgrowth by a calmodulin antagonism in N1E-115 cells. Recently, direct in vitro activation of protein kinase C by melatonin was described. Vimentin intermediate filaments are attached to microtubules and their organization depends on both microtubule distribution and phosphorylation of specific proteins. Protein kinase C is a serine threonine kinase which phosphorylates vimentin and through this mechanism causes intermediate filament disassembly. In this work the effects of melatonin on protein kinase C activation, content, and subcellular distribution were studied in N1E-115 cells. Also, melatonin effects on vimentin phosphorylation and subcellular distribution were explored. The results show that melatonin both activates and increases protein kinase C content in the membrane cytoskeletal fraction. Melatonin protein kinase C activation was followed by an increase in both vimentin phosphorylation and by vimentin subcellular redistribution. Moreover, staurosporine, a serine threonine kinase inhibitor, prevented increased vimentin phosphorylation elicited by melatonin. Similar effects to those caused by melatonin were obtained with the protein kinase C activator phorbol 12-myristate 13-acetate. Data support the idea that melatonin modulates vimentin organization through protein kinase C activation.     

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

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 patient's prostate cancer. As indicated by his PSA levels, melatonin induced stabilization of his hormone-refractory disease for 6 wk. This report validates melatonin's 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.     

Biotransformation of melatonin in human breast cancer cell lines: role of sulfotransferase 1A1

The biologically active melatonin metabolite, 6-hydroxymelatonin (6-OHMel), is conjugated to form 6-hydroxymelatonin sulfate (6-OHMelS). To elucidate the role of the sulfotransferase (SULT) enzyme 1A1, considerably expressed in normal and malignant human breast cells, we measured the formation of 6-OHMelS by ELISA in hormone-dependent MCF-7 and hormone-independent MDA-MB231 (MDA) breast cancer cell lines after stable transfection with SULT1A1. In parent MDA cells, low SULT1A1 mRNA expression was associated with moderate 6-OHMelS formation as determined after application (24 hr) of 0.1 microM 6-OHMel. As expected, overexpression of SULT1A1 in MDA cells resulted in a 2.9- and 110-fold increase in 6-OHMelS in the cytosol and cellular supernatant respectively. Furthermore, 6.3- and 115-fold increases were observed after 0.5 microM, and 12.6- and 101-fold increases after 1 microM 6-OHMel respectively. In MCF-7 cells, because of high basal SULT1A1 expression, only two- to threefold increases in 6-OHMelS were observed after transfection with the enzyme. In total, 866 and 539 pmol/mg protein 6-OHMelS were formed from 1 microM 6-OHMel in SULT1A1 overexpressing MDA and MCF-7 cells, respectively, whereas application of 1 microM melatonin produced only <1% of 6-OHMelS. Possible interactions with the SULT1A1 substrate tamoxifen (tam), an anti-estrogen applied in the therapy of breast cancer, were also studied. A concentration of 1 microM tam increased 6-OHMelS formation by approximately threefold in the presence of 1 microM melatonin or 1 microM 6-OHMel respectively. However, no alterations were detected after application of 1 microM 4-hydroxy-tamoxifen. In summary, we demonstrate the importance of SULT1A1 for the biotransformation of 6-OHMel in human breast cancer cells. Our data further suggest that tam can modulate melatonin biotransformation.     

Synergistic effect of melatonin and ghrelin in preventing cisplatin‐induced ovarian damage via regulation of FOXO3a phosphorylation and binding to the p27Kip1 promoter in primordial follicles

Premature ovarian failure during chemotherapy is a serious problem for young women with cancer. To preserve the fertility of these patients, approaches to prevent chemotherapy‐induced ovarian failure are needed. In a previous study, we reported that melatonin treatment prevents the depletion of the dormant follicle pool via repression of the simultaneous activation of dormant primordial follicles by cisplatin. However, melatonin's protective effect was only partial and thus insufficient. In this study, we found that the hormone ghrelin enhances the protective effect of melatonin against cisplatin‐induced ovarian failure in mouse model. Co‐administration of melatonin and ghrelin more effectively prevented cisplatin‐induced follicle disruption. Simultaneous treatment with melatonin and ghrelin almost restored the number of primordial follicles and the corpus luteum in cisplatin‐treated ovaries, compared with single administration. We found melatonin and ghrelin receptors on the cell membrane of premature oocytes of primordial follicles. In addition, melatonin and ghrelin co‐administration inhibited the cisplatin‐induced phosphorylation of PTEN and FOXO3a that induces cytoplasmic translocation of FOXO3a. Inhibition of FOXO3a phosphorylation by melatonin and ghrelin increased the binding affinity of FOXO3a for the p27^Kip1 promoter in primordial follicles. Co‐administration of melatonin and ghrelin in cisplatin‐treated ovaries restored the expression of p27^Kip1, which is critical for retention of the dormant status of primordial follicles. In conclusion, these findings suggest that melatonin and ghrelin co‐administration is suitable for use as a fertoprotective adjuvant therapy during cisplatin chemotherapy in young female cancer patients.     

Expression of melatonin receptor MT1 in cells of human invasive ductal breast carcinoma

In humans, two main types of membrane melatonin receptors have been identified, MT1 and MT2. Expression of MT1 in neoplastic cells seems to increase the efficacy of melatonin's oncostatic activity. The purpose of this study was to determine the distribution and the intensity of MT1 expression in breast cancer cells and to correlate it with clinicopathological factors. Immunohistochemical studies (IHC) were conducted on 190 cases of invasive ductal breast carcinomas (IDC) and molecular studies were performed on 29 cases of frozen tumor fragments and selected breast cancer cell lines. Most of the studied tumors manifested a membranous/cytoplasmic IHC expression of MT1. In IDC, the MT1 expression was higher than in fibrocystic breast disease. MT1 expression was higher in estrogen receptor positive (ER+) and HER2 positive (HER2+) tumors. Triple negative tumors (TN) manifested the lowest MT1 expression level. The lowest MT1 protein expression level was noted in the TN breast cancer cell line MDA‐MB‐231 compared with ER+ cell lines MCF‐7 and SK‐BR‐3. MT1 mRNA expression was negatively correlated with the malignancy grade of the studied IDC cases. Moreover, higher MT1 expression was associated with patients' longer overall survival (OS) in the group of ER+ breast cancers and treated with tamoxifen. Multivariate analysis indicated that MT1 was an independent prognostic factor in the ER+ tumors for OS and event‐free survival in the ER+ tumors. The results of this study may point to a potential prognostic and therapeutic significance of MT1 in IDC.     

Cytoplasmic expression of p27(kip1) is associated with a favourable prognosis in colorectal cancer patients

INTRODUCTION p27, also known as Kip1, is expressed in most cells and its role is to bind and inhibit cyclin/cyclin-dependent kinase (cyclin-CDK) complexes, thereby inhibiting cell cycle progression[1]. This role in the cell cycle makes p27kip1 a key playe…     

A1蛋白与前列腺特异性抗原在前列腺癌与前列腺增生疾病中联合检测的意义

目的选取50~70岁之间的前列腺增生、前列腺癌患者和健康对照者,对其血液中A1蛋白的表达与PSA的表达水平进行比较分析,为前列腺增生和前列腺癌的临床鉴别诊断提供依据。方法采用电化学发光仪检测PSA浓度,采用反转录酶-聚合酶链锁反应(RT-PCR)技术和蛋白免疫印迹法(western-blot)检测20例前列腺癌患者、20例前列腺增生患者和20例正常者新鲜前列腺组织中A1 mRNA和蛋白的表达情况,探讨A1蛋白表达与前列腺增生和前列腺癌中PSA表达水平的关系。结果 RT-PCR与westem-blot检测结果一致,A1蛋白在前列腺增生组织和前列腺癌组织中均有阳性表达,前列腺癌组织中A1蛋白阳性表达明显高于前列腺增生组织,有显著性差异(P0.01)。A1蛋白表达的阳性率与PSA的浓度相关。结论 A1蛋白阳性表达程度与前列腺病变程度相关,A1蛋白在细胞凋亡调节中起着重要的作用,其过度表达在前列腺癌的发生、发展中扮演着重要的角色。联合检测A1和PSA的表达变化对判断前列腺癌的生物学行为有参考价值。     

胰腺癌p27kip1、视网膜母细胞瘤基因蛋白和增殖细胞核抗原表达及与临床病理关系的研究

目的　探讨细胞周期负性调控因子p2 7kip1,视网膜母细胞瘤 (Rb)基因蛋白和增殖细胞核抗原 (PC NA)在胰腺癌发生、发展中的作用。方法　应用免疫组织化学技术 (SP法 ) ,对 32例胰腺癌及癌旁组织中p2 7kip1、Rb蛋白和PCNA表达进行检测。结果　p2 7kip1蛋白阳性表达率在胰腺癌组织中为 5 6 2 5 %,显著低于癌旁胰腺组织 (84 37%) (P <0 0 5 ) ,并与胰腺癌组织分化程度及淋巴结转移相关 (P <0 0 5 ) ;Rb基因蛋白阳性表达率在胰腺癌组织中为 5 0 0 0 %,显著低于癌旁胰腺组织 (78 13%) (P <0 0 5 ) ;PCNA阳性表达率在胰腺癌组织中为 71 87%,显著高于癌旁胰腺组织 (4 3 75 %) (P <0 0 5 ) ,并与胰腺癌组织分化程度和淋巴结转移均相关(P <0 .0 5 )。结论　p2 7kip1、Rb基因蛋白和PCNA与胰腺癌发生、发展密切相关。     

Molecular deficiency (ies) in MT1 melatonin signaling pathway underlies the melatonin‐unresponsive phenotype in MDA‐MB‐231 human breast cancer cells

Melatonin has been shown repeatedly to inhibit the growth of human breast tumor cells in vitro and in vivo. Its antiproliferative effects have been well studied in MCF‐7 human breast cancer cells and several other estrogen receptor α (ERα)‐positive human breast cancer cell lines. However, the MDA‐MB‐231 breast cancer cell line, an ERα‐negative cell line widely used in breast cancer research, has been shown to be unresponsive to melatonin's growth‐suppressive effect in vitro. Here, we examined the effect of melatonin on the cell proliferation of several ERα‐negative breast cancer cell lines including MDA‐MB‐231, BT‐20, and SK‐BR‐3 cells. Although the MT1 G‐protein‐coupled receptor is expressed in all three cell lines, melatonin significantly suppressed the proliferation of SK‐BR‐3 cells without having any significant effect on the growth of MDA‐MB‐231 and BT‐20 cells. We confirmed that the MT1‐associated Gα proteins are expressed in MDA‐MB‐231 cells. Further studies demonstrated that the melatonin unresponsiveness in MDA‐MB‐231 cells may be caused by aberrant signaling downstream of the Gαi proteins, resulting in differential regulation of ERK1/2 activity.     

Melatonin decreases cell proliferation and induces melanogenesis in human melanoma SK‐MEL‐1 cells

Abstract: Melatonin is an indoleamine synthesized in the pineal gland, and after its release into the blood, it has an extensive repertoire of biological activities, including antitumoral properties. In this study, we found that melatonin reduced the growth of the human melanoma cells SK‐MEL‐1. The antiproliferative effect was associated with an alteration in the progression of the phases of the cell cycle and also with an increase in tyrosinase activity, the key regulatory enzyme of melanogenesis. Antagonists for melatonin membrane receptors (luzindole and 4‐P‐PDOT) and the general G‐coupled receptor inhibitor, pertussis toxin, did not prevent the melatonin‐induced cell growth arrest; this suggests a mechanism independent of G‐coupled membrane receptors. In contrast, p38 mitogen‐activated protein kinase (p38 MAPK) signaling pathway seems to play a significant role in cell growth inhibition by melatonin. The indoleamine‐induced phosphorylation of p38 MAPK and the effect on cell proliferation were abrogated by the specific inhibitor SB203580 . Furthermore, comparative studies with known antioxidants such as N‐acetyl‐l ‐cysteine and trolox indicate that the growth of SK‐MEL‐1 cells is highly sensitive to antioxidants.     

Melatonin induces apoptotic death in LNCaP cells via p38 and JNK pathways: therapeutic implications for prostate cancer

Abstract:  Reactive oxygen species (ROS) are involved in pathophysiology of ischemia/reperfusion injury. Melatonin is a potent scavenger of ROS. Thus, this study was designed to elucidate its effects in a combined hepatic warm ischemia and resection model. The right lateral and caudate lobes (32% of liver volume) of Sprague–Dawley rats underwent warm ischemia for 30 min followed by reperfusion and subsequent resection of the nonischemic liver tissue. Some rats were gavaged with 50 mg/kg melatonin 2 hr before the onset of experiments. Controls received the same volume of microcrystalline cellulose. Survival, transaminases, histology, flow cytometry, inducible nitric oxide synthase (iNOS) expression, and activation of signal transduction pathways [c-Jun N-terminal kinase (JNK), cJUN, IκB kinase α (IKKα), proliferating cell nuclear antigen (PCNA), and Ki67] were assessed for hepatic injury, oxidative stress, and cell proliferation. Melatonin significantly improved animal survival and decreased transaminase levels, the indices for necrosis, liver damage, leukocyte infiltration, and iNOS expression. In parallel, the expression of IKKα, JNK1, and cJUN decreased by 35–50% after melatonin ( P  <   0.05). At the same time, melatonin reduced the expression of both PCNA and Ki67 in liver ( P  <   0.05). Melatonin is hepatoprotective most likely via mechanisms including inhibition of IKK and JNK pathways and regulation of cell proliferation.     

Antitumour activity of melatonin in a mouse model of human prostate cancer: relationship with hypoxia signalling

Melatonin is known to exert antitumour activity in several types of human cancers, but the underlying mechanisms as well as the efficacy of different doses of melatonin are not well defined. Here, we test the hypothesis whether melatonin in the nanomolar range is effective in exerting antitumour activity in vivo and examine the correlation with the hypoxia signalling mechanism, which may be a major molecular mechanism by which melatonin antagonizes cancer. To test this hypothesis, LNCaP human prostate cancer cells were xenografted into seven‐wk‐old Foxn1nu/nu male mice that were treated with melatonin (18 i.p. injections of 1 mg/kg in 41 days). Saline‐treated mice served as control. We found that the melatonin levels in plasma and xenografted tissue were 4× and 60× higher, respectively, than in control samples. Melatonin tended to restore the redox imbalance by increasing expression of Nrf2. As part of the phenotypic response to these perturbations, xenograft microvessel density was less in melatonin‐treated animals, indicative of lower angiogenesis, and the xenograft growth rate was slower (P < 0.0001). These changes were accompanied by a reduced expression of Ki67, elevated expression of HIF‐1α and increased phosphorylation of Akt in melatonin than saline‐treated mice. We conclude that the beneficial effect of melatonin in reducing cancer growth in vivo was evident at melatonin plasma levels as low as 4 nm and was associated with decreased angiogenesis. Higher HIF‐1α expression in xenograft tissue indicates that the antitumour effect cannot be due to a postulated antihypoxic effect, but may stem from lower angiogenesis potential.     

Intracrine androgen metabolism in prostate cancer progression: mechanisms of castration resistance and therapeutic implications

Residual tissue androgens are consistently detected within the prostate tumors of castrate individuals and are thought to play a critical role in facilitating the androgen receptor-mediated signaling pathways leading to disease progression. The source of residual tumor androgens is attributed in part to the uptake and conversion of circulating adrenal androgens. Whether the de novo biosynthesis of androgens from cholesterol or earlier precursors occurs within prostatic tumors is not known, but it has significant implications for treatment strategies targeting sources of androgens exogenous to the prostate versus 'intracrine' sources within the prostatic tumor. Moreover, increased expression of androgen-metabolizing genes within castration-resistant metastases suggests that up-regulated activity of endogenous steroidogenic pathways may contribute to the outgrowth of 'castration-adapted' tumors. These observations suggest that a multi-targeted treatment approach designed to simultaneously ablate testicular, adrenal and intracrine contributions to the tumor androgen signaling axis will be required to achieve optimal therapeutic efficacy.