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.     

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.     

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.     

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 attenuates neuronal apoptosis through up‐regulation of K+–Cl− cotransporter KCC2 expression following traumatic brain injury in rats

Traumatic brain injury (TBI) initiates a complex cascade of neurochemical and signaling changes that leads to neuronal apoptosis, which contributes to poor outcomes for patients with TBI. The neuron‐specific K⁺–Cl^? cotransporter‐2 (KCC2), the principal Cl^? extruder in adult neurons, plays an important role in Cl^? homeostasis and neuronal function. This present study was designed to investigate the expression pattern of KCC2 following TBI and to evaluate whether or not melatonin is able to prevent neuronal apoptosis by modulating KCC2 expression in a Sprague Dawley rat controlled cortical impact model of TBI. The time course study showed decreased mRNA and protein expression of KCC2 in the ipsilateral peri‐core parietal cortex after TBI. Double immunofluorescence staining demonstrated that KCC2 is located in the plasma membrane of neurons. In addition, melatonin (10 mg/kg) was injected intraperitoneally at 5 minutes and repeated at 1, 2, 3, and 4 hours after brain trauma, and brain samples were extracted 24 hours after TBI. Compared to the vehicle group, melatonin treatment altered the down‐regulation of KCC2 expression in both mRNA and protein levels after TBI. Also, melatonin treatment increased the protein levels of brain‐derived neurotrophic factor (BDNF) and phosphorylated extracellular signal‐regulated kinase (p‐ERK). Simultaneously, melatonin administration ameliorated cortical neuronal apoptosis, reduced brain edema, and attenuated neurological deficits after TBI. In conclusion, our findings suggested that melatonin restores KCC2 expression, inhibits neuronal apoptosis and attenuates secondary brain injury after TBI, partially through activation of BDNF/ERK pathway.     

Inhibiting MT2‐TFE3‐dependent autophagy enhances melatonin‐induced apoptosis in tongue squamous cell carcinoma

Autophagy modulation is a potential therapeutic strategy for tongue squamous cell carcinoma (TSCC). Melatonin possesses significant anticarcinogenic activity. However, whether melatonin induces autophagy and its roles in cell death in TSCC are unclear. Herein, we show that melatonin induced significant apoptosis in the TSCC cell line Cal27. Apart from the induction of apoptosis, we demonstrated that melatonin‐induced autophagic flux in Cal27 cells as evidenced by the formation of GFP‐LC3 puncta, and the upregulation of LC3‐II and downregulation of SQSTM1/P62. Moreover, pharmacological or genetic blockage of autophagy enhanced melatonin‐induced apoptosis, indicating a cytoprotective role of autophagy in melatonin‐treated Cal27 cells. Mechanistically, melatonin induced TFE3^(Ser321) dephosphorylation, subsequently activated TFE3 nuclear translocation, and increased TFE3 reporter activity, which contributed to the expression of autophagy‐related genes and lysosomal biogenesis. Luzindole, a melatonin membrane receptor blocker, or MT2‐siRNA partially blocked the ability of melatonin to promote mTORC1/TFE3 signaling. Furthermore, we verified in a xenograft mouse model that melatonin with hydroxychloroquine or TFE3‐siRNA exerted a synergistic antitumor effect by inhibiting autophagy. Importantly, TFE3 expression positively correlated with TSCC development and poor prognosis in patients. Collectively, we demonstrated that the melatonin‐induced increase in TFE3‐dependent autophagy is mediated through the melatonin membrane receptor in TSCC. These data also suggest that blocking melatonin membrane receptor‐TFE3‐dependent autophagy to enhance the activity of melatonin warrants further attention as a treatment strategy for TSCC.     

Melatonin blunts the mitochondrial/NLRP3 connection and protects against radiation‐induced oral mucositis

Mucositis is a common and distressing side effect of chemotherapy or radiotherapy that has potentially severe consequences, and no treatment is available. The purpose of this study was to analyze the molecular pathways involved in the development of oral mucositis and to evaluate whether melatonin can prevent this pathology. The tongue of male Wistar rats was subjected to irradiation (X‐ray YXLON Y.Tu 320‐D03 irradiator; the animals received a dose of 7.5 Gy/day for 5 days). Rats were treated with 45 mg/day melatonin or vehicle for 21 days postirradiation, either by local application into their mouths (melatonin gel) or by subcutaneous injection. A connection between reactive oxygen species, generating mitochondria and the NLRP3 (NLR‐related protein 3 nucleotide‐binding domain leucine‐rich repeat containing receptor‐related protein 3) inflammasome, has been reported in mucositis. Here, we show that mitochondrial oxidative stress, bioenergetic impairment and subsequent NLRP3 inflammasome activation are involved in the development of oral mucositis after irradiation and that melatonin synthesized in the rat tongue is depleted after irradiation. The application of melatonin gel restores physiological melatonin levels in the tongue and prevents mucosal disruption and ulcer formation. Melatonin gel protects the mitochondria from radiation damage and blunts the NF‐κB/NLRP3 inflammasome signaling activation in the tongue. Our results suggest new molecular pathways involved in radiotherapy‐induced mucositis that are inhibited by topical melatonin application, suggesting a potential preventive therapy for mucositis in patients with cancer.     

Dexamethasone attenuates methacholine‐mediated aquaporin 5 downregulation in human nasal epithelial cells via suppression of NF‐κB activation

Background

Cholinergic stimulation plays a major role in inflammatory airway diseases. However, its role in airway surface liquid homeostasis and aquaporin 5 (AQP5) regulation remains unclear. In this study we sought to determine the effects of methacholine and dexamethasone on AQP5 expression in human nasal epithelial cells (HNEpC).

Methods

HNEpC were cultured with methacholine or dexamethasone at 4 concentrations in vitro. The subcellular distribution of AQP5 was explored using immunocytochemistry. The pharmacologic effects of methacholine and dexamethasone on the expression of the phosphorylation of cyclic adenosine monophosphate–responsive element binding protein (p‐CREB), AQP5, and nuclear factor‐kappaB (NF‐κB) were examined using Western blotting.

Results

AQP5 was found to be located in cell membrane and cytoplasm and present in every group without a statistically significant difference. Methacholine inhibited expression of AQP5 and p‐CREB in HNEpC, whereas dexamethasone increased these protein levels dose‐dependently in a statistically significant manner. In turn, HNEpC treated with methacholine and dexamethasone showed the same trends as those intervened separately with these 2 drugs. Moreover, dexamethasone had the ability to reverse the inhibitory effect of methacholine. Western blotting revealed that, after incubation with 10^?4 mol/L methacholine, NF‐κB increased significantly, by 186.67%, compared with the untreated control group. Again, such an increase could be significantly reversed after dexamethasone treatment.

Conclusion

NF‐κB activation is important for inhibition of p‐CREB/AQP5 expression after methacholine intervention, and dexamethasone adjusts it to the opposite side. This observation could provide additional insight into the anti‐inflammatory effects of glucocorticoids that contribute to maintaining airway surface liquid and mucosal defense.

     

Monocytic differentiation of leukemic HL‐60 cells induced by co‐treatment with TNF‐α and MK886 requires activation of pro‐apoptotic machinery

The block of hematopoietic differentiation program in acute myeloid leukemia cells can be overcome by differentiating agent like retinoic acid, but it has several side effects. A study of other differentiation signaling pathways is therefore useful to predict potential targets of anti‐leukemic therapy. We demonstrated previously that the co‐treatment of HL‐60 cells with Tumor necrosis factor‐α (TNF‐α) (1 ng/mL) and inhibitor of 5‐lipoxygenase MK886 (5 μm ) potentiated both monocytic differentiation and apoptosis. In this study, we detected enhanced activation of three main types of mitogen‐activated protein kinases (MAPKs) (p38, c‐Jun amino‐terminal kinase [JNK], extracellular signal‐regulated kinase [ERK]), so we assessed their role in differentiation using appropriate pharmacologic inhibitors. The inhibition of pro‐apoptotic MAPKs (p38 and JNK) suppressed the effect of MK886 + TNF‐α co‐treatment. On the other hand, down‐regulation of pro‐survival ERK pathway led to increased differentiation. Those effects were accompanied by increased activation of caspases in cells treated by MK886 + TNF‐α. Pan‐caspase inhibitor ZVAD‐fmk significantly decreased both number of apoptotic and differentiated cells. The same effect was observed after inhibition of caspase 9, but not caspase 3 and 8. To conclude, we evidenced that the activation of apoptotic processes and pathways supporting apoptosis (p38 and JNK MAPKs) is required for the monocytic differentiation of HL‐60 cells.