Melatonin overcomes gemcitabine resistance in pancreatic ductal adenocarcinoma by abrogating nuclear factor‐κB activation

Constitutive activation and gemcitabine induction of nuclear factor‐κB (NF‐κB) contribute to the aggressive behavior and chemotherapeutic resistance of pancreatic ductal adenocarcinoma (PDAC). Thus, targeting the NF‐κB pathway has proven an insurmountable challenge for PDAC therapy. In this study, we investigated whether the inhibition of NF‐κB signaling pathway by melatonin might lead to tumor suppression and overcome gemcitabine resistance in pancreatic tumors. Our results showed that melatonin inhibited activities of NF‐κB by suppressing IκBα phosphorylation and decreased the expression of NF‐κB response genes in MiaPaCa‐2, AsPc‐1, Panc‐28 cells and gemcitabine resistance MiaPaCa‐2/GR cells. Moreover, melatonin not only inhibited cell proliferation and invasion in a receptor‐independent manner, but also enhanced gemcitabine cytotoxicity at pharmacologic concentrations in these PDAC cells. In vivo, the mice treated with both agents experienced a larger reduction in tumor burden than the single drug‐treated groups in an orthotopic xenograft mouse model. Taken together, these results indicate that melatonin inhibits proliferation and invasion of PDAC cells and overcomes gemcitabine resistance of pancreatic tumors through NF‐κB inhibition. Our findings therefore provide novel preclinical knowledge about melatonin inhibition of NF‐κB in PDAC and suggest that melatonin should be investigated clinically, alone or in combination with gemcitabine for PDAC treatment.     

Increased synovial tissue NF‐κB1 expression at sites adjacent to the cartilage–pannus junction in rheumatoid arthritis

Objective

To compare the expression of the Rel/NF‐κB subunits, NF‐κB1 (p50) and RelA (p65), in paired synovial tissue samples selected from sites adjacent to and remote from the cartilage–pannus junction (CPJ) in patients with inflammatory arthritis.

Methods

Synovial tissue was selected at arthroscopy from sites adjacent to the CPJ and from the suprapatellar pouch of patients who were referred to an early arthritis clinic. Tissue samples from patients with osteoarthritis (OA) undergoing knee arthroplasty were also studied. Rel/NF‐κB subunit activation and expression were measured by electrophoretic mobility shift assay and supershift analyses and by immunohistochemistry.

Results

Tissue samples were obtained from 10 patients with rheumatoid arthritis (RA), 7 with a seronegative arthropathy (SnA), and 6 with OA. Rel/NF‐κB was abundantly expressed in all samples. In both RA and SnA synovial tissue, the absolute number of NF‐κB1+ cells at the CPJ was significantly higher than at non‐CPJ sites (P = 0.006 and P = 0.02, respectively). The proportion of cells expressing NF‐κB1 was also significantly higher at the CPJ compared with non‐CPJ sites (P = 0.003 in RA, P = 0.009 in SnA). The numbers of RelA+ cells were consistently lower throughout. In RA synovial tissue, but not in SnA synovial tissue, both the absolute number and the proportion of RelA+ cells were significantly higher at the CPJ than at non‐CPJ sites (P = 0.003 and P = 0.01, respectively). In OA synovial tissue, the numbers of cells expressing NF‐κB1 and RelA were similar to those observed at the non‐CPJ sites in all inflammatory tissues studied.

Conclusion

In this study of early inflammatory arthritis, expression of NF‐κB1 in synovial tissue was highest at sites most likely to be associated with joint erosion. These observations are consistent with a critical role of NF‐κB1 in joint destruction, and support the rationale for specific therapeutic inhibition of NF‐κB in RA.

     

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.     

Clinicopathological significance of nuclear factor‐κB activation in hepatocellular carcinoma

Aim: Nuclear factor‐κB (NF‐κB) is a critical signaling mediator in inflammation, apoptosis resistance and oncogenesis. It has been reported that NF‐κB is activated in several cancers, including hepatocellular carcinoma (HCC). Studies of genetic disruptions in mice also suggest that NF‐κB plays critical roles in hepatocarcinogenesis. The aim of the present study is to characterize NF‐κB activation and correlate it with the degree of malignancy in HCC. Methods: To examine the correlation between the positivity of the nuclear p50 subunit and HCC recurrence, we analyzed immunostaining of the NF‐κB p50 subunit in two groups of HCC samples with known prognosis and Akt phosphorylation status: 49 patients showing early recurrence within 6 months (group A) and 50 patients who were recurrence‐free for at least for 3 years (group B). Results: In group A, positive nuclear staining of p50 was shown in 18 cases (36.7%), whereas only one case (2.0%) in group B had positive nuclear staining of p50 (P = 2.48839 × 10^–5). This suggests a positive relationship between nuclear p50 and early recurrence and advanced HCC in humans. The presence of phosphorylated Akt correlated with nuclear staining of p50 in HCCs in group A (R² = 0.213, P < 0.001). Conclusion: Our results indicate that nuclear staining of p50 was clearly associated with early recurrent HCC, and the Akt pathway might play a role in NF‐κB activation in a subset of early recurrent HCC.     

Melatonin synergizes the chemotherapeutic effect of 5‐fluorouracil in colon cancer by suppressing PI3K/AKT and NF‐κB/iNOS signaling pathways

5‐Fluorouracil (5‐FU) is one of the most commonly used chemotherapeutic agents in colon cancer treatment, but has a narrow therapeutic index limited by its toxicity. Melatonin exerts antitumor activity in various cancers, but it has never been combined with 5‐FU as an anticolon cancer treatment to improve the chemotherapeutic effect of 5‐FU. In this study, we assessed such combinational use in colon cancer and investigated whether melatonin could synergize the antitumor effect of 5‐FU. We found that melatonin significantly enhanced the 5‐FU‐mediated inhibition of cell proliferation, colony formation, cell migration and invasion in colon cancer cells. We also found that melatonin synergized with 5‐FU to promote the activation of the caspase/PARP‐dependent apoptosis pathway and induce cell cycle arrest. Further mechanism study demonstrated that melatonin synergized the antitumor effect of 5‐FU by targeting the PI3K/AKT and NF‐κB/inducible nitric oxide synthase (iNOS) signaling. Melatonin in combination with 5‐FU markedly suppressed the phosphorylation of PI3K, AKT, IKKα, IκBα, and p65 proteins, promoted the translocation of NF‐κB p50/p65 from the nuclei to cytoplasm, abrogated their binding to the iNOS promoter, and thereby enhanced the inhibition of iNOS signaling. In addition, pretreatment with a PI3K‐ or iNOS‐specific inhibitor synergized the antitumor effects of 5‐FU and melatonin. Finally, we verified in a xenograft mouse model that melatonin and 5‐FU exerted synergistic antitumor effect by inhibiting the AKT and iNOS signaling pathways. Collectively, our study demonstrated that melatonin synergized the chemotherapeutic effect of 5‐FU in colon cancer through simultaneous suppression of multiple signaling pathways.     

Melatonin inhibits the sphingosine kinase 1/sphingosine‐1‐phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin

The sphingosine kinase (SphK)1/sphingosine‐1‐phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV‐infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll‐like receptor (TLR)4, tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6, nuclear factor‐kappa B (NF‐κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.     

Nuclear factor‐κB as a potential therapeutic target for the novel cytotoxic agent LC‐1 in acute myeloid leukaemia

Nuclear factor‐κB (NF‐κB) has been implicated in a number of malignancies and has been suggested to be a potential molecular target in the treatment of leukaemia. This study demonstrated the constitutive activation of NF‐κB in human myeloid blasts and a clear correlation between NF‐κB expression and in vitro cytoprotection. High NF‐κB expression was found in many of the poor prognostic acute myeloid leukaemia (AML) subtypes, such as French‐American‐British classification M0 and M7, and the poor cytogenetic risk group. The in vitro effects of LC‐1, a novel dimethylamino‐parthenolide analogue, were assessed in 62 primary untreated AML samples. LC‐1 was found to be cytotoxic to AML cells in a dose‐dependent manner, mediated through the induction of apoptosis. The median drug concentration necessary to kill 50% of the cells was 4·5 μmol/l for AML cells, compared with 12·8 μmol/l for normal marrow cells. LC‐1 was shown to reduce the five individual human NF‐κB Rel proteins in a dose‐dependent manner. The subsequent inhibition of many NF‐κB‐regulated cytokines was also demonstrated. Importantly, sensitivity to LC‐1 was correlated with the basal NF‐κB activity. Consequently, LC‐1 treatment provides a proof of principle for the use of NF‐κB inhibitors in the treatment of AML.