Melatonin induces apoptosis of colorectal cancer cells through HDAC4 nuclear import mediated by CaMKII inactivation

Melatonin induces apoptosis in many different cancer cell lines, including colorectal cancer. However, the precise mechanisms involved remain largely unresolved. In this study, we provide evidence to reveal a new mechanism by which melatonin induces apoptosis of colorectal cancer LoVo cells. Melatonin at pharmacological concentrations significantly suppressed cell proliferation and induced apoptosis in a dose‐dependent manner. The observed apoptosis was accompanied by the melatonin‐induced dephosphorylation and nuclear import of histone deacetylase 4 (HDAC4). Pretreatment with a HDAC4‐specific siRNA effectively attenuated the melatonin‐induced apoptosis, indicating that nuclear localization of HDAC4 is required for melatonin‐induced apoptosis. Moreover, constitutively active Ca²⁺/calmodulin‐dependent protein kinase II alpha (CaMKIIα) abrogated the melatonin‐induced HDAC4 nuclear import and apoptosis of LoVo cells. Furthermore, melatonin decreased H3 acetylation on bcl‐2 promoter, leading to a reduction of bcl‐2 expression, whereas constitutively active CaMKIIα(T286D) or HDAC4‐specific siRNA abrogated the effect of melatonin. In conclusion, the present study provides evidence that melatonin‐induced apoptosis in colorectal cancer LoVo cells largely depends on the nuclear import of HDAC4 and subsequent H3 deacetylation via the inactivation of CaMKIIα.     

Melatonin enhances hyperthermia‐induced apoptotic cell death in human leukemia cells

Melatonin is an endogenous indoleamine with a wide range of biological functions. In addition to modulating circadian rhythms, it plays important roles in the health as an antioxidant. Melatonin has also the ability to induce apoptosis in cancer cells and to enhance the antitumoral activity of chemotherapeutic agents. In this study, the effect of melatonin on hyperthermia‐induced apoptosis was explored using human leukemia cells. The results demonstrate that melatonin greatly improved the cytotoxicity of hyperthermia in U937 cells. The potentiation of cell death was achieved with 1 mmol/L concentrations of the indoleamine but not with concentrations close to physiological levels in blood (1 nmol/L). This effect was associated to an enhancement of the apoptotic response, revealed by an increase in cells with hypodiploid DNA content and activation of multiple caspases (caspase‐2, caspase‐3, caspase‐8, and caspase‐9). Melatonin also increased hyperthermia‐induced Bid activation as well as translocation of Bax from the cytosol to mitochondria and cytochrome c release. Hyperthermia‐provoked apoptosis and potentiation by melatonin were abrogated by a broad‐spectrum caspase inhibitor (z‐VAD‐fmk) as well as by specific inhibitors against caspase‐8 or caspase‐3. In contrast, blocking of the mitochondrial pathway of apoptosis either with a caspase‐9 inhibitor or overexpressing the anti‐apoptotic protein Bcl‐2 (U937/Bcl‐2) reduced the number of apoptotic cells in response to hyperthermia but it was unable to suppress melatonin enhancement. Melatonin appears to modulate the apoptotic response triggered by hyperthermia in a cell type‐specific manner as similar results were observed in HL‐60 but not in K562 or MOLT‐3 cells.     

Melatonin inhibits cell proliferation and induces caspase activation and apoptosis in human malignant lymphoid cell lines

Abstract: Melatonin exerts strong anti‐tumour activity via several mechanisms, including anti‐proliferative and pro‐apoptotic effects in addition to its potent antioxidant activity. Several studies have investigated the effects of melatonin on haematological malignancies. However, the previous studies investigating lymphoid malignancies have been largely restricted to a single type of malignancy, Burkitt’s lymphoma (BL). Thus, we examined the actions of melatonin on the growth and apoptosis in a small panel of cell lines representing different human lymphoid malignancies including Ramos (Epstein–Barr virus–negative BL), SU‐DHL‐4 (diffuse large B cell lymphoma), DoHH2 (follicular B non‐Hodgkin lymphoma) and JURKAT (acute T cell leukaemia). We showed that melatonin promotes cell cycle arrest and apoptosis in all these cells, although there was marked variations in responses among different cell lines (sensitivity; Ramos/DoHH2 > SU‐DHL‐4 > JURKAT). Melatonin‐induced apoptosis was relatively rapid, with increased caspase 3 and PARP cleavage detected within 0.5–1 h following melatonin addition. Moreover, there was evidence for rapid processing of both caspase 9, as well as a breakdown of the mitochondrial inner transmembrane potential. On the contrary, caspase activation was detected only in SU‐DHL‐4 and Ramos cells following melatonin treatment suggesting that the extrinsic pathway does not make a consistent contribution to melatonin‐induced apoptosis in malignant lymphocytes. Although all cell lines expressed the high‐affinity melatonin receptors, MT1 and MT2, melatonin‐induced caspase activation appeared to be independent these receptors. Our findings confirm that melatonin could be a potential chemotherapeutic/preventive agent for malignant lymphocytes. However, it is necessary to take into account that different lymphoid malignancies may differ in their response to melatonin.     

Melatonin reverses flow shear stress‐induced injury in bone marrow mesenchymal stem cells via activation of AMP‐activated protein kinase signaling

Tissue‐engineered heart valves (TEHVs) are a promising treatment for valvular heart disease, although their application is limited by high flow shear stress (FSS). Melatonin has a wide range of physiological functions and is currently under clinical investigation for expanded applications; moreover, extensive protective effects on the cardiovascular system have been reported. In this study, we investigated the protection conferred by melatonin supplementation against FSS‐induced injury in bone marrow mesenchymal stem cells (BMSCs) and elucidated the potential mechanism in this process. Melatonin markedly reduced BMSC apoptotic death in a concentration‐dependent manner while increasing the levels of transforming growth factor β (TGF‐β), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF) and B‐cell lymphoma 2 (Bcl2), and decreasing those of Bcl‐2‐associated X protein (Bax), p53 upregulated modulator of apoptosis (PUMA), and caspase 3. Notably, melatonin exerted its protective effects by upregulating the phosphorylation of adenosine monophosphate‐activated protein kinase (AMPK), which promotes acetyl‐CoA carboxylase (ACC) phosphorylation. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked the anti‐FSS injury (anti‐FSSI) effects of melatonin. Inhibition of AMPK by Compound C also counteracted the protective effects of melatonin, suggesting that melatonin reverses FSSI in BMSCs through the AMPK‐dependent pathway. Overall, our findings indicate that melatonin contributes to the amelioration of FSS‐induced BMSC injury by activating melatonin receptors and AMPK/ACC signaling. Our findings may provide a basis for the design of more effective strategies that promote the use of TEHCs in patients.     

Long noncoding RNA H19 mediates melatonin inhibition of premature senescence of c‐kit+ cardiac progenitor cells by promoting miR‐675

Melatonin, a hormone secreted by the pineal gland, possesses multiple biological activities such as antitumor, antioxidant, and anti‐ischemia. C‐kit⁺ cardiac progenitor cells (CPCs) have emerged as a promising tool for the treatment of heart diseases. However, the senescence of CPCs due to pathological stimuli leads to the decline of CPCs' functions and regenerative potential. This study was conducted to demonstrate whether melatonin antagonizes the senescence of CPCs in response to oxidative stress. Here, we found that the melatonin treatment markedly inhibited the senescent characteristics of CPCs after exposed to sublethal concentration of H₂O₂, including the increase in senescence‐associated β‐galactosidase (SA‐β‐gal)‐positive CPCs, senescence‐associated heterochromatin loci (SAHF), secretory IL‐6 level, and the upregulation of p53 and p21 proteins. Senescence‐associated proliferation reduction was also attenuated by melatonin in CPCs. Luzindole, the melatonin membrane receptor blocker, may block the melatonin‐mediated suppression of premature senescence in CPCs. Interestingly, we found that long noncoding RNA H19 and its derived miR‐675 were downregulated by H₂O₂ in CPCs, but melatonin treatment could counter this alteration. Furthermore, knockdown of H19 or miR‐675 blocked antisenescence actions of melatonin on H₂O₂‐treated CPCs. It was further verified that H19‐derived miR‐675 targeted at the 3′UTR of USP10, which resulted in the downregulation of p53 and p21 proteins. In summary, melatonin antagonized premature senescence of CPCs via H19/miR‐675/USP10 pathway, which provides new insights into pharmacological actions and potential applications of melatonin on the senescence of CPCs.     

Melatonin reduces indomethacin‐induced gastric mucosal cell apoptosis by preventing mitochondrial oxidative stress and the activation of mitochondrial pathway of apoptosis

Abstract: Augmentation of gastric mucosal cell apoptosis due to development of oxidative stress is one of the main pathogenic events in the development of nonsteroidal anti‐inflammatory drug (NSAID)‐induced gastropathy. Identification of a nontoxic, anti‐apoptotic molecule is warranted for therapy against NSAID‐induced gastropathy. The objective of the present study was to define the mechanism of the anti‐apoptotic effect of melatonin, a nontoxic molecule which scavenges reactive oxygen species. Using an array of experimental approaches, we have shown that melatonin prevents the development of mitochondrial oxidative stress and activation of mitochondrial pathway of apoptosis induced by indomethacin (a NSAID) in the gastric mucosa. Melatonin inhibits the important steps of indomethacin‐induced activation of mitochondrial pathway of apoptosis such as upregulation of the expression of Bax and Bak, and the downregulation of Bcl‐2 and BclxL. Melatonin also prevents indomethacin‐induced mitochondrial translocation of Bax and prevents the collapse of mitochondrial membrane potential. Moreover, melatonin reduces indomethacin‐mediated activation of caspase‐9 and caspase‐3 by blocking the release of cytochrome c and finally rescues gastric mucosal cells from indomethacin‐induced apoptosis as measured by the TUNEL assay. Histologic studies of gastric mucosa further document that melatonin almost completely protects against gastric damage induced by indomethacin. Thus, melatonin has significant anti‐apoptotic effects to protect gastric mucosa from NSAID‐induced apoptosis and gastropathy, which makes its use as potential therapy against gastric damage during NSAID treatment.     

Melatonin attenuated early brain injury induced by subarachnoid hemorrhage via regulating NLRP3 inflammasome and apoptosis signaling

Subarachnoid hemorrhage (SAH) is a devastating condition with high morbidity and mortality rates due to the lack of effective therapy. Nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome activation associated with the upregulation of apoptotic signaling pathway has been implicated in various inflammatory diseases including hemorrhagic insults. Melatonin is reported to possess substantial anti‐inflammatory properties, which is beneficial for early brain injury (EBI) after SAH. However, the molecular mechanisms have not been clearly identified. This study was designed to investigate the protective effects of melatonin against EBI induced by SAH and to elucidate the potential mechanisms. The adult mice were subjected to SAH. Melatonin or vehicle was injected intraperitoneally 2 hr after SAH. Melatonin was neuroprotective, as shown by increased survival rate, as well as elevated neurological score, greater survival of neurons, preserved brain glutathione levels, and reduced brain edema, malondialdehyde concentrations, apoptotic ratio, and blood–brain barrier (BBB) disruption. Melatonin also attenuated the expressions of NLRP3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), cleaved caspase‐1, interleukin‐1β (IL‐1β), and interleukin‐6 (IL‐6); these changes were also associated with an increase in the anti‐apoptotic factor (Bcl2) and reduction in the pro‐apoptotic factor (Bim). In summary, our results demonstrate that melatonin treatment attenuates the EBI following SAH by inhibiting NLRP3 inflammasome‐associated apoptosis.     

Melatonin attenuates hypoxic pulmonary hypertension by inhibiting the inflammation and the proliferation of pulmonary arterial smooth muscle cells

Hypoxia‐induced inflammation and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Melatonin possesses anti‐inflammatory and antiproliferative properties. However, the effect of melatonin on HPH remains unclear. In this study, adult Sprague–Dawley rats were exposed to intermittent chronic hypoxia for 4 wk to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio, and median width of pulmonary arterioles. Melatonin attenuated the elevation of RVSP, RV/LV+S, and mitigated the pulmonary vascular structure remodeling. Melatonin also suppressed the hypoxia‐induced high expression of proliferating cell nuclear antigen (PCNA), hypoxia‐inducible factor‐1α (HIF‐1α), and nuclear factor‐κB (NF‐κB). In vitro, melatonin concentration‐dependently inhibited the proliferation of PASMCs and the levels of phosphorylation of Akt and extracellular signal‐regulated kinases1/2 (ERK1/2) caused by hypoxia. These results suggested that melatonin might potentially prevent HPH via anti‐inflammatory and antiproliferative mechanisms.     

Melatonin improves reprogramming efficiency and proliferation of bovine‐induced pluripotent stem cells

Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC‐derived pluripotent stem cells (N‐iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N‐iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR‐302/367‐mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N‐iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis‐related genes p53 and p21. Then, N‐iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N‐iPS (M‐N‐iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N‐iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M‐N‐iPS via melatonin receptors 1 (MT1). Finally, we verified that N‐iPS cells and M‐N‐iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers.     

Evaluation of the immunomodulatory effect of melatonin on the T‐cell response in peripheral blood from systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of antinuclear autoantibodies. In addition, the involvement of CD4+ T‐helper (Th) cells in SLE has become increasingly evident. Although the role of melatonin has been tested in some experimental models of lupus with inconclusive results, there are no studies evaluating the melatonin effect on cells from patients with SLE. Therefore, the aim of this study was to analyse the role of in vitro administered melatonin in the immune response of peripheral leukocytes from treated patients with SLE (n = 20) and age‐ and sex‐matched healthy controls. Melatonin was tested for its effect on the production of key Th1, Th2, Th9, Th17 and innate cytokines. The frequency of T regulatory (Treg) cells and the expression of FOXP3 and BAFF were also explored. Our results are the first to show that melatonin decreased the production of IL‐5 and to describe the novel role of melatonin in IL‐9 production by human circulating cells. Additionally, we highlighted a two‐faceted melatonin effect. Although it acted as a prototypical anti‐inflammatory compound, reducing exacerbated Th1 and innate responses in PHA‐stimulated cells from healthy subjects, it caused the opposite actions in immune‐depressed cells from patients with SLE. Melatonin also increased the number of Treg cells expressing FOXP3 and offset BAFF overexpression in SLE patient cells. These findings open a new field of research in lupus that could lead to the use of melatonin as treatment or cotreatment for SLE.     

Melatonin prevents pancreatic β‐cell loss due to glucotoxicity: the relationship between oxidative stress and endoplasmic reticulum stress

Prolonged hyperglycemia results in pancreatic β‐cell dysfunction and apoptosis, referred to as glucotoxicity. Although both oxidative and endoplasmic reticulum (ER) stresses have been implicated as major causative mechanisms of β‐cell glucotoxicity, the reciprocal importance between the two remains to be elucidated. The aim of this study was to evaluate the differential effect of oxidative stress and ER stress on β‐cell glucotoxicity, by employing melatonin which has free radical‐scavenging and antioxidant properties. As expected, in β‐cells exposed to prolonged high glucose levels, cell viability and glucose‐stimulated insulin secretion (GSIS) were significantly impaired. Melatonin treatment markedly attenuated cellular apoptosis by scavenging reactive oxygen species via its plasmalemmal receptor‐independent increase in antioxidant enzyme activity. However, treatments with antioxidants alone were insufficient to recover the impaired GSIS. Interestingly, 4‐phenylbutyric acid (4‐PBA), a chemical chaperone that attenuate ER stress by stabilizing protein structure, alleviated the impaired GSIS, but not apoptosis, suggesting that glucotoxicity induces oxidative and ER stress independently. We found that cotreatment of glucotoxic β‐cells with melatonin and 4‐PBA dramatically improved both their survival and insulin secretion. Taken together, these results suggest that ER stress may be the more critical mechanism for prolonged high‐glucose‐induced GSIS impairment, whereas oxidative stress appears to be more critical for the impaired β‐cell viability. Therefore, combinatorial therapy of melatonin with an ER stress modifier may help recover pancreatic β‐cells under glucotoxic conditions in type 2 diabetes.     

Immunoregulatory actions of melatonin and zinc during chronic Trypanosoma cruzi infection

After one century of the discovery of Chagas' disease and the development of an efficient drug with amplitude of actions both in the acute and chronic phase is still a challenge. Alternative immune modulators have been exhaustively used. For that purpose, melatonin and zinc were administered during chronic Trypanosoma cruzi‐infected Wistar rats and several endpoints were assessed. Melatonin has a remarkable functional versatility, being associated with important antioxidant, anti‐inflammatory, and anti‐apoptotic effects. The cross‐talk between zinc and the immune system includes its ability to influence the production and signaling of numerous inflammatory cytokines in a variety of cell types. Our study showed that zinc triggered a decrease in the generation of IFN‐γ for TCD4⁺ cells. Reduced percentage of CD4⁺T cells producing TNF‐α was observed in control melatonin or zinc‐and‐melatonin‐treated animals as compared with untreated rats. On the other hand, a significant increase in the percentage of IL‐4 from CD4⁺ and CD8⁺ T lymphocytes producers was observed 60 days after infection, for all zinc‐treated animals, whether infected or not. Melatonin and zinc therapies increased the percentages of CD4⁺ and CD8⁺ T lymphocytes IL‐10 producers. CD4⁺CD25^highFoxp3⁺ T cells were also elevated in zinc‐ and melatonin‐treated animals. The modulation of the immune system influenced by these molecules affected cytokine production and the inflammatory process during chronic T. cruzi infection. Elucidation of the interplay between cytokine balance and the pathogenesis of Chagas’ disease is extremely relevant not only for the comprehension of the immune mechanisms and clinical forms but, most importantly, also for the implementation of efficient and adequate therapies.     

Melatonin significantly improves the developmental competence of bovine somatic cell nuclear transfer embryos

Somatic cell nuclear transfer (SCNT) is a promising technology, but its application is hampered by its low efficiency. Hence, the majority of SCNT embryos fail to develop to term. In this study, the antioxidant melatonin reduced apoptosis and reactive oxygen species (ROS) in bovine SCNT embryos. It also increased cell number, inner cell mass (ICM) cell numbers, and the ratio of ICM to total cells while improving the development of bovine SCNT embryos in vitro and in vivo. Gene expression analysis showed that melatonin suppressed the expression of the pro‐apoptotic genes p53 and Bax and stimulated the expression of the antioxidant genes SOD1 and Gpx4, the anti‐apoptotic gene BCL2L1, and the pluripotency‐related gene SOX2 in SCNT blastocysts. We also analyzed the epigenetic modifications in bovine in vitro fertilization, melatonin‐treated, and untreated SCNT embryos. The global H3K9ac levels of melatonin‐treated SCNT embryos at the four‐cell stage were higher than those of the untreated SCNT embryos. We conclude that exogenous melatonin affects the expression of genes related to apoptosis, antioxidant function, and development. Moreover, melatonin reduced apoptosis and ROS in bovine SCNT embryos and enhanced blastocyst quality, thereby ultimately improving bovine cloning efficiency.     

Melatonin and its derivatives counteract the ultraviolet B radiation‐induced damage in human and porcine skin ex vivo

Melatonin and its derivatives (N¹‐acetyl‐N²‐formyl‐5‐methoxykynurenine [AFMK] and N‐acetyl serotonin [NAS]) have broad‐spectrum protective effects against photocarcinogenesis, including both direct and indirect antioxidative actions, regulation of apoptosis and DNA damage repair; these data were primarily derived from in vitro models. This study evaluates possible beneficial effects of melatonin and its active derivatives against ultraviolet B (UVB)‐induced harm to human and porcine skin ex vivo and to cultured HaCaT cells. The topical application of melatonin, AFMK, or NAS protected epidermal cells against UVB‐induced 8‐OHdG formation and apoptosis with a further increase in p53^ser15 expression, especially after application of melatonin or AFMK but not after NAS use. The photoprotective action was observed in pre‐ and post‐UVB treatment in both human and porcine models. Melatonin along with its derivatives upregulated also the expression of antioxidative enzymes after UVB radiation of HaCaT cells. The exogenous application of melatonin or its derivatives represents a potent and promising tool for preventing UVB‐induced oxidative stress and DNA damage. This protection results in improved genomic, cellular, and tissue integrity against UVB‐induced carcinogenesis, especially when applied prior to UV exposure. In addition, our ex vivo experiments provide fundamental justification for further testing the clinical utility of melatonin and metabolites as protectors again UVB in human subjects. Our ex vivo data constitute the bridge between vitro to vivo translation and thus justifies the pursue for further clinical utility of melatonin in maintaining skin homeostasis.     

Melatonin inhibits cholangiocarcinoma and reduces liver injury in Opisthorchis viverrini‐infected and N‐nitrosodimethylamine‐treated hamsters

The human liver fluke Opisthorchis viverrini infection and N‐nitrosodimethylamine (NDMA) administration induce cholangiocarcinoma (CCA) and liver injury in hamsters. Melatonin protects against liver injury and reduces the alteration of mitochondrial structure, mitochondrial membrane potential, and mitochondrial pro‐ and anti‐apoptotic pathways in various cancer types. To investigate the chemopreventive effect of melatonin on CCA genesis and liver injury, hamsters were treated with a combination of O. viverrini infection and NDMA concurrently administered with melatonin (10 mg/kg and 50 mg/kg) for 120 days. Melatonin treatment at 50 mg/kg caused a significant reduction in liver/body weight ratios and decreased tumor volumes leading to an increase in the survival of animals. In the tumorous tissues, the high‐dose melatonin reduced DNA fragmentation and mitochondrial apoptosis by inducing anti‐apoptotic protein (Bcl‐2) in the mitochondrial fraction and down‐regulating cytochrome c, pro‐apoptotic protein (Bax), and caspase‐3 in tumor cytosol. Moreover, a high‐dose melatonin treatment significantly increased mitochondrial antioxidant enzymes and prevented mitochondrial ultrastructure changes in the tumor. Overall, melatonin has potent chemopreventive effects in inhibiting CCA genesis and also reduces liver injury in hamster CCA, which, in part, might involve in the suppression of CCA by reducing tumor mitochondria alteration.     

Melatonin regulates the calcium‐buffering proteins,parvalbumin and hippocalcin,in ischemic brain injury

Abstract: Melatonin has anti‐oxidant activity and it exerts a neuroprotective effects during ischemic brain injury. Calcium‐buffering proteins including parvalbumin and hippocalcin are involved in neuronal differentiation and maturation through calcium signaling. This study investigated whether melatonin moderates parvalbumin and hippocalcin expression in cerebral ischemia and glutamate toxicity‐induced neuronal cell death. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Male Sprague‐Dawley rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortical tissues were collected 24 hr after MCAO. Parvalbumin and hippocalcin levels were decreased in vehicle‐treated animal with MCAO, whereas melatonin prevented the ischemic injury‐induced reduction in these proteins. In cultured hippocampal cells, glutamate toxicity decreased parvalbumin and hippocalcin levels, while melatonin treatment prevented the glutamate exposure‐induced diminished in these proteins levels. Melatonin also attenuated the glutamate toxicity‐induced increase in intracellular Ca²⁺ levels. These results suggest that the maintenance of parvalbumin and hippocalcin levels by melatonin in ischemic injury contributes to the neuroprotective effect of melatonin against neuronal cell damage.     

Melatonin sensitizes human breast cancer cells to ionizing radiation by downregulating proteins involved in double‐strand DNA break repair

Radiation and adjuvant endocrine therapy are nowadays considered a standard treatment option after surgery in breast cancer. Melatonin exerts oncostatic actions on human breast cancer cells. In the current study, we investigated the effects of a combination of radiotherapy and melatonin on human breast cancer cells. Melatonin (1 mm , 10 μm and 1 nm ) significantly inhibited the proliferation of MCF‐7 cells. Radiation alone inhibited the MCF‐7 cell proliferation in a dose‐dependent manner. Pretreatment of breast cancer cells with melatonin 1 wk before radiation led to a significantly greater decrease of MCF‐7 cell proliferation compared with radiation alone. Melatonin pretreatment before radiation also decreased G₂‐M phase arrest compared with irradiation alone, with a higher percentage of cells in the G₀‐G₁ phase and a lower percentage of cells in S phase. Radiation alone diminished RAD51 and DNA‐protein kinase (PKcs) mRNA expression, two main proteins involved in double‐strand DNA break repair. Treatment with melatonin for 7 days before radiation led to a significantly greater decrease in RAD51 and DNA?PKcs mRNA expression compared with radiation alone. Our findings suggest that melatonin pretreatment before radiation sensitizes breast cancer cells to the ionizing effects of radiation by decreasing cell proliferation, inducing cell cycle arrest and downregulating proteins involved in double‐strand DNA break repair. These findings may have implications for designing clinical trials using melatonin and radiotherapy.