Melatonin attenuates TNF‐α and IL‐1β expression in synovial fibroblasts and diminishes cartilage degradation: Implications for the treatment of rheumatoid arthritis

The hormone melatonin has many properties, including antioxidant, anti‐inflammatory, and immunomodulatory effects. Melatonin has been demonstrated to be beneficial in several inflammatory autoimmune diseases, but its effects in rheumatoid arthritis (RA) remain controversial. We sought to determine how melatonin regulates inflammation in RA. We found that melatonin dose‐dependently inhibits tumor necrosis factor‐α (TNF‐α) and interleukin (IL)‐1β expression through the PI3K/AKT, ERK, and NF‐κB signaling pathways. We also identified that melatonin inhibits TNF‐α and IL‐1β production by upregulating miR‐3150a‐3p expression. Synovial tissue specimens from RA patients and culture of human rheumatoid fibroblast‐like synoviocytes confirmed that the MT1 receptor is needed for the anti‐inflammatory activities of melatonin. Importantly, melatonin also significantly reduced paw swelling, cartilage degradation, and bone erosion in the collagen‐induced arthritis mouse model. Our results indicate that melatonin ameliorates RA by inhibiting TNF‐α and IL‐1β production through downregulation of the PI3K/AKT, ERK, NF‐κB signaling pathways, as well as miR‐3150a‐3p overexpression. The role of melatonin as an adjuvant treatment in patients with RA deserves further clinical studies.     

Melatonin potentiates the antiproliferative and pro‐apoptotic effects of ursolic acid in colon cancer cells by modulating multiple signaling pathways

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is largely distributed in medical herbs and edible plants. Melatonin is an indoleamine compound produced in the pineal gland and also a plant‐derived product. Both UA and melatonin have been shown to inhibit cancer cell growth in numerous studies, but they have never been combined altogether as an anticolon cancer treatment. In this study, we investigated whether the association between UA and melatonin leads to an enhanced antiproliferative and pro‐apoptotic activities in colon cancer SW480 and LoVo cells. We found that combined treatment with UA and melatonin significantly enhanced inhibition of cell viability and migration, promoted changes in cell morphology and spreading, and increased induction of apoptosis, thereby potentiating the effects of UA alone in colon cancer cells. Moreover, we found that the enhanced effects of UA and melatonin combination are mediated through simultaneous modulation of cytochrome c/caspase, MMP9/COX‐2, and p300/NF‐κB signaling pathways. Combined treatment with UA and melatonin triggered the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, induced cleavage of caspase and PARP proteins, enhanced inhibition of MMP9 and COX‐2 expression, promoted p300 and NF‐κB translocation from cell nuclei to cytoplasm, and abrogated NF‐κB binding and p300 recruitment to COX‐2 promoter in colon cancer cells. These results, therefore, demonstrated that melatonin potentiated the antiproliferative and pro‐apoptotic effects of UA in colon cancer cells by modulating multiple signaling pathways and suggest that such a combinational treatment might potentially become an effective way in colon cancer therapy.     

Curcumin attenuates high glucose‐induced inflammatory injury through the reactive oxygen species–phosphoinositide 3‐kinase/protein kinase B–nuclear factor‐κB signaling pathway in rat thoracic aorta endothelial cells

Aims/Introduction

Endothelial cell inflammatory injury is likely required for barrier dysfunction under hyperglycemic conditions. Curcumin (CUR) is well known for its anti‐inflammatory effect. However, there have been few reports about the anti‐inflammatory effect of CUR induced by high glucose in endothelial cells. The aim of the present study was to investigate the inflammatory effect of high glucose and the anti‐inflammatory effect of CUR induced by high glucose in rat thoracic aorta endothelial cells (TAECs).

Materials and Methods

Well characterized TAECs were established and cell viability was assayed by the cell counting kit‐8 method, messenger ribonucleic acid and protein expression were identified by real‐time polymerase chain reaction, western blot or enzyme‐linked immunosorbent assay, respectively. The production of reactive oxygen species was observed by a fluorescence microscope.

Results

High glucose (30 mmol/L) significantly decreased the cell viability of TAECs after being co‐cultivated for 12 h and showed a time‐dependent manner, and increased interleukin (IL)‐1β, IL‐6 and tumor necrosis factor‐α secretion in TAECs. The injury effect of high glucose was involved in the reactive oxygen species–phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT)–nuclear factor (NF)‐κB signaling pathway. Anti‐oxidant N‐acetylcysteine, PI3K and NF‐κB‐specific pathway inhibitors can abolish the secretion of these inflammatory factors; pretreatment with anti‐oxidant N‐acetylcysteine significantly decreased PI3K expression, the level of phosphorylated AKT and nuclear NF‐κB; pretreatment of LY294002 can significantly decrease the NF‐κB level in nuclei. After treatment with CUR for 12 h, IL‐1β, IL‐6 and tumor necrosis factor‐α secretion were markedly decreased, and PI3K expression, the phosphorylation of AKT and nuclear NF‐κB level were also decreased.

Conclusion

Curcumin attenuates high glucose‐induced inflammatory injury through the reactive oxygen species–PI3K/AKT–NF‐κB signaling pathway in rat thoracic aorta endothelial cells.     

Melatonin ameliorates Aβ42‐induced alteration of βAPP‐processing secretases via the melatonin receptor through the Pin1/GSK3β/NF‐κB pathway in SH‐SY5Y cells

Melatonin is involved in the physiological regulation of the β‐amyloid precursor protein (βAPP)‐cleaving secretases which are responsible for generation of the neurotoxic amyloid beta (Aβ) peptide, one of the hallmarks of Alzheimer's disease (AD) pathology. In this study, we aimed to determine the underlying mechanisms of this regulation under pathological conditions. We establish that melatonin prevents Aβ₄₂‐induced downregulation of a disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) as well as upregulation of β‐site APP‐cleaving enzyme 1 (BACE1) and presenilin 1 (PS1) in SH‐SY5Y cell cultures. We also demonstrate that the intrinsic mechanisms of the observed effects occurred via regulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and glycogen synthase kinase (GSK)‐3β as melatonin reversed Aβ₄₂‐induced upregulation and nuclear translocation of NF‐κBp65 as well as activation of GSK3β via its receptor activation. Furthermore, specific blocking of the NF‐κB and GSK3β pathways partially abrogated the Aβ₄₂‐induced reduction in the BACE1 and PS1 levels. In addition, GSK3β blockage affected α‐secretase cleavage and modulated nuclear translocation of NF‐κB. Importantly, our study for the first time shows that peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 (Pin1) is a crucial target of melatonin. The compromised levels and/or genetic variation of Pin1 are associated with age‐dependent tau and Aβ pathologies and neuronal degeneration. Interestingly, melatonin alleviated the Aβ₄₂‐induced reduction of nuclear Pin1 levels and preserved the functional integrity of this isomerase. Our findings illustrate that melatonin attenuates Aβ₄₂‐induced alterations of βAPP‐cleaving secretases possibly via the Pin1/GSK3β/NF‐κB pathway.     

Immunohistochemical study of nuclear factor‐κB expression in esophageal squamous cell carcinoma: prognostic significance and sensitivity to treatment with 5‐FU

Nuclear factor‐κB (NF‐κB) is expressed in many types of cancers. It has been suggested that the expression of NF‐κB is associated with poor prognosis and resistance to chemoradiation therapies. This study evaluated the relationship between the expression of NF‐κB and the prognosis and sensitivity of esophageal squamous cell carcinoma (ESCC) to chemotherapy. One hundred and nine ESCC specimens, from patients who had undergone radical esophagectomy, were divided into two groups depending on the expression of NF‐κB. Surgical data and prognosis were compared between the two groups. NF‐κB‐positive tumors were detected in 61.5% of the cases. In 69 patients with stage II and III disease, 41 patients who were NF‐κB‐positive showed poor survival. The sensitivity of esophageal squamous cell carcinoma cell lines to 5‐fluorouracil (5‐FU) was analyzed by their NF‐κB expression, and the effect of 5‐FU was evaluated on the proliferation and activity of two cell lines of cultured ESCCs expressing NF‐κB. ESCCs with activated NF‐κB had poor sensitivity to 5‐FU. These results suggest that the increased expression of NF‐κB is associated with poor prognosis in patients with ESCC. NF‐κB may be a target for ESCC therapy because of its selective expression in this type of cancer.     

Melatonin prevents deregulation of the sphingosine kinase/sphingosine 1‐phosphate signaling pathway in a mouse model of diethylnitrosamine‐induced hepatocellular carcinoma

The sphingosine kinase (SphK)/sphingosine 1‐phosphate (S1P) pathway is involved in multiple biological processes, including carcinogenesis. Melatonin shows beneficial effects in cell and animal models of hepatocellular carcinoma, but it is unknown if they are associated with the modulation of the SphK/S1P system, along with different downstream signaling pathways modified in cancer. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight i.p) once a week for 8 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p. beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Melatonin alleviated the distortion of normal hepatic architecture, lowered the incidence of preneoplastic/neoplastic lesions, and inhibited the expression of proliferative/cell cycle regulatory proteins (Ki67, PCNA, cyclin D1, cyclin E, CDK4, and CDK6). S1P levels and expression of SphK1, SphK2, and S1P receptors (S1PR1/S1PR3) were significantly elevated in DEN‐treated mice. However, there was a decreased expression of S1P lyase. These effects were significantly abrogated in a time‐ and dose‐dependent manner by melatonin, which also increased S1PR2 expression. Following DEN treatment, mice exhibited increased phosphorylation of PI3K, AKT, mTOR, STAT3, ERK, and p38, and a higher expression of NF‐κB p50 and p65 subunits. Melatonin administration significantly inhibited those changes. Data obtained suggest a contribution of the SphK/S1P system and related signaling pathways to the protective effects of melatonin in hepatocarcinogenesis.     

Melatonin down‐regulates MDM2 gene expression and enhances p53 acetylation in MCF‐7 cells

Compelling evidence demonstrated that melatonin increases p53 activity in cancer cells. p53 undergoes acetylation to be stabilized and activated for driving cells destined for apoptosis/growth inhibition. Over‐expression of p300 induces p53 acetylation, leading to cell growth arrest by increasing p21 expression. In turn, p53 activation is mainly regulated in the nucleus by MDM2. MDM2 also acts as E3 ubiquitin ligase, promoting the proteasome‐dependent p53 degradation. MDM2 entry into the nucleus is finely tuned by two different modulations: the ribosomal protein L11, acts by sequestering MDM2 in the cytosol, whereas the PI3K‐AkT‐dependent MDM2 phosphorylation is mandatory for MDM2 translocation across the nuclear membrane. In addition, MDM2‐dependent targeting of p53 is regulated in a nonlinear fashion by MDM2/MDMX interplay. Melatonin induces both cell growth inhibition and apoptosis in MCF7 breast cancer cells. We previously reported that this effect is associated with reduced MDM2 levels and increased p53 activity. Herein, we demonstrated that melatonin drastically down‐regulates MDM2 gene expression and inhibits MDM2 shuttling into the nucleus, given that melatonin increases L11 and inhibits Akt‐PI3K‐dependent MDM2 phosphorylation. Melatonin induces a 3‐fold increase in both MDMX and p300 levels, decreasing simultaneously Sirt1, a specific inhibitor of p300 activity. Consequently, melatonin‐treated cells display significantly higher values of both p53 and acetylated p53. Thus, a 15‐fold increase in p21 levels was observed in melatonin‐treated cancer cells. Our results provide evidence that melatonin enhances p53 acetylation by modulating the MDM2/MDMX/p300 pathway, disclosing new insights for understanding its anticancer effect.     

Melatonin ameliorates cerulein‐induced pancreatitis by the modulation of nuclear erythroid 2‐related factor 2 and nuclear factor‐kappaB in rats

Abstract: Melatonin exhibits a wide variety of biological effects, including antioxidant and anti‐inflammatory functions. Its antioxidant role impedes the etiopathogenesis of pancreatitis, but little is known about the signaling pathway of melatonin in the induction of antioxidant enzymes in acute pancreatitis (AP). The aim of this study was to determine whether melatonin could prevent cerulein‐induced AP through nuclear factor erythroid 2‐related factor 2 (Nrf2) and curtail inflammation by inhibition of NF‐κB. AP was induced by two intraperitoneal (i.p.) injections of cerulein at 2 h intervals (50 μg/kg) in Sprague‐Dawley rats. Melatonin (10 or 50 mg/kg/daily, i.p.) was administered 24 h before each injection of cerulein. The rats were killed 12 h after the last injection. Acinar cell degeneration, pancreatic edema, and inflammatory infiltration were significantly different in cerulein‐ and melatonin‐treated rats. Melatonin significantly reduced amylase, lipase, MPO, and MDA levels, and increased antioxidant enzyme activities including SOD and GPx, which were decreased in AP (P < 0.05). Melatonin increased the expression of NQO1, HO‐1, and SOD2 when compared with the cerulein‐induced AP group (P < 0.05). In addition, melatonin increased Nrf2 expression, and reduced expressions of tumor necrosis factor‐alpha, IL‐1β, IL‐6, IL‐8, and iNOS. The elevated nuclear binding of NF‐κB in the cerulein‐induced pancreatitis group was inhibited by melatonin. These results show that melatonin increases antioxidant enzymes and Nrf2 expression, and limits inflammatory mediators in cerulein‐induced AP. It is proposed that melatonin may play an important role in oxidative stress via the Nrf2 pathway in parallel with reduction of inflammation by NF‐κB inhibition.     

Inhibition of nuclear factor κB and phosphatidylinositol 3‐kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor α in mice

Background/aims: Tumor necrosis factor (TNF)‐α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF‐α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)‐κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF‐α. Here, we focused on the roles of NF‐κB and phosphatidylinositol 3‐kinase (PI3K)‐regulated serine/threonine kinase Akt. Methods: TNF‐α was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IκBα (Ad5IκB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl‐2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5IκB or the wortmannin suppressed the activation of NF‐κB or Akt, respectively. Suppression of either NF‐κB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF‐α administration. However, in mice treated with both Ad5IκB and wortmannin, TNF‐α administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IκB, wortmannin, and TNF‐α markedly increased the activation of caspase‐3 and ‐9, and activated caspase‐8 to a lesser degree, suggesting that TNF‐α‐induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF‐κB and PI3K/Akt pathways had no effect on expression of Bcl‐2 families. Conclusion: The inducible activation of NF‐κB and constitutive activation of Akt regulate hepatocyte survival against TNF‐α, which occurs independent of Bcl‐2 families.     

Melatonin modulates neuroinflammation and oxidative stress in experimental diabetic neuropathy: effects on NF-κB and Nrf2 cascades

Abstract: Melatonin exhibits an array of biological activities, including antioxidant and anti‐inflammatory actions. Diabetic neuropathy is one of the complications of diabetes with a prevalence rate of 50–60%. We have previously reported the protective effect of melatonin in experimental diabetic neuropathy. In this study, we investigated the role of nuclear factor‐kappa B (NF‐κB) and nuclear erythroid 2‐related factor 2 (Nrf2) in melatonin‐mediated protection against streptozotocin‐induced diabetic neuropathy. Melatonin at doses of 3 and 10 mg/kg was administered daily in seventh and eighth week after diabetes induction. Motor nerve conduction velocity and nerve blood flow were improved in melatonin‐treated animals. Melatonin also reduced the elevated expression of NF‐κB, IκB‐α, and phosphorylated IκB‐α. Further, melatonin treatment also reduced the elevated levels of proinflammatory cytokines (TNF‐α and IL‐6), iNOS and COX‐2 in sciatic nerves of animals. The capacity of melatonin to modulate Nrf2 pathway was associated with increased heme oxygenase‐1 (HO‐1) expression, which strengthens antioxidant defense. This fact was also established by decreased DNA fragmentation (because inhibition of excessive oxidant‐induced DNA damage) in the sciatic nerve of melatonin‐treated animals. The results of this study suggest that melatonin modulates neuroinflammation by decreasing NF‐κB activation cascade and oxidative stress by increasing Nrf2 expression, which might be responsible at least in part, for its neuroprotective effect in diabetic neuropathy.     

Melatonin sensitizes human cervical cancer HeLa cells to cisplatin‐induced cytotoxicity and apoptosis: effects on oxidative stress and DNA fragmentation

Melatonin has antitumor activity via several mechanisms including its antiproliferative and pro‐apoptotic effects as well as its potent antioxidant actions, although recent evidence has indicated that melatonin may perform pro‐oxidant actions in tumor cells. Therefore, melatonin may be useful in the treatment of tumors in association with chemotherapy drugs. This study was intended to evaluate the in vitro effect of melatonin on the cytotoxic and pro‐apoptotic actions of various chemotherapeutic agents in cervical cancer HeLa cells. Herein, we found that both melatonin and three of the chemotherapeutic drugs tested, namely cisplatin (CIS), 5‐fluorouracil (5‐FU), and doxorubicin, induced a decrease in HeLa cell viability. Furthermore, melatonin significantly increased the cytotoxic effect of such chemotherapeutic agents. Consistently, costimulation of HeLa cells with any chemotherapeutic agent in the presence of melatonin further increased caspase‐3 activation, particularly in CIS‐ and 5‐FU‐challenged cells. Likewise, concomitant treatments with melatonin and CIS significantly enhanced the ratio of cells entering mitochondrial apoptosis due to reactive oxygen species (ROS) overproduction, substantially augmented the population of apoptotic cells, and markedly enlarged DNA fragmentation compared to the treatments with CIS alone. Nonetheless, melatonin only displayed moderate chemosensitizing effects in 5‐FU‐stimulated HeLa cells, as suggested by slight increments in the percentage of cells stimulated for ROS production and in the proportion of early apoptotic cells compared to the treatments with 5‐FU alone. In summary, our findings provided evidence that in vitro melatonin strongly enhances CIS‐induced cytotoxicity and apoptosis in HeLa cells and, hence, the indoleamine could be potentially applied to cervical cancer treatment as a powerful synergistic agent.     

Melatonin modulates TLR4‐mediated inflammatory genes through MyD88‐ and TRIF‐dependent signaling pathways in lipopolysaccharide‐stimulated RAW264.7 cells

Abstract: Increasing evidence demonstrates that melatonin has an anti‐inflammatory effect. Nevertheless, the molecular mechanisms remain obscure. In this study, we investigated the effect of melatonin on toll‐like receptor 4 (TLR4)‐mediated molecule myeloid differentiation factor 88 (MyD88)‐dependent and TRIF‐dependent signaling pathways in lipopolysaccharide (LPS)‐stimulated macrophages. RAW264.7 cells were incubated with LPS (2.0 μg/mL) in the absence or presence of melatonin (10, 100, 1000 μm ). As expected, melatonin inhibited TLR4‐mediated tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1β, IL‐6, IL‐8, and IL‐10 in LPS‐stimulated macrophages. In addition, melatonin significantly attenuated LPS‐induced upregulation of cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) in macrophages. Further analysis showed that melatonin inhibited the expression of MyD88 in LPS‐stimulated macrophages. Although it had no effect on TLR4‐mediated phosphorylation of c‐Jun N‐terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK), melatonin significantly attenuated the activation of nuclear factor kappa B (NF‐κB) in LPS‐stimulated macrophages. In addition, melatonin inhibited TLR4‐mediated Akt phosphorylation in LPS‐stimulated macrophages. Moreover, melatonin significantly attenuated the elevation of interferon (IFN)‐regulated factor‐3 (IRF3), which was involved in TLR4‐mediated TRIF‐dependent signaling pathway, in LPS‐stimulated macrophages. Correspondingly, melatonin significantly alleviated LPS‐induced IFN‐β in macrophages. In conclusion, melatonin modulates TLR4‐mediated inflammatory genes through MyD88‐dependent and TRIF‐dependent signaling pathways.