Melatonin has been shown to be produced by nonpineal cells and possess anti-inflammatory actions in animal models. In the present study, we tested the hypothesis that melatonin suppresses the expression of proinflammatory genes such as cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (INOS) by a common transcriptional mechanism. Melatonin but not tryptophan or serotonin inhibited lipopolysaccharide (LPS)-induced COX-2 and iNOS protein levels and promoter activities in RAW 264.7 cells in a time-and concentration-dependent manner. LPS or LPS plus interferon-␥ (IFN␥) increased binding of all 5 isoforms of NF-B to COX-2 and iNOS promoters. Melatonin selectively inhibited p52 binding without affecting p100 expression, p52 generation from p100, or p52 nuclear translocation. p52 acetylation was enhanced by LPS, which was abrogated by melatonin. Melatonin inhibited p300 histone acetyltransferase (HAT) activity and abrogated p300-augmented COX-2 and iNOS expression. HAT inhibitors suppressed LPS-induced p52 binding and acetylation to an extent similar to melatonin, and melatonin did not potentiate the effect of HAT inhibitors. These results suggest that melatonin inhibits COX-2 and iNOS transcriptional activation by inhibiting p300 HAT activity, thereby suppressing p52 acetylation, binding, and transactivation. ( IntroductionMelatonin is produced primarily in the pineal gland and secreted into circulating blood. It has been shown that melatonin is also produced by cells including monocytes and macrophages and has been implicated in protection against inflammation (for a review, see Reiter 1 ). Depletion of endogenous melatonin has been shown in rats to aggravate carrageenan-induced pleurisy, which is reversed by exogenous melatonin, 2 while exogenous melatonin administration has been demonstrated to reduce acute inflammatory reactions in zymosan-activated plasma-induced paw inflammation 3 and carrageenan-induced edema and pleurisy. 4 Its anti-inflammatory actions are attributed to its ability to scavenge reactive oxygen species and suppress proinflammatory genes, including inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX2). 5,6 iNOS (also known as NOS-2) and COX-2 play an important role in diverse inflammatory conditions including vascular inflammation, atherosclerosis, and plaque stability. 7-10 iNOS catalyzes the robust synthesis of nitric oxide (NO) from L-arginine. 11 NO induces inflammatory reactions and tissue injury by diverse mechanisms including activation of soluble guanylyl cyclase, protein nitration, and nitrosylation. 12-14 COX-2 occupies a key position in production of proinflammatory prostanoids such as prostaglandin E 2 (PGE 2 ). 15 iNOS and COX-2 share similar cellular and molecular properties. 16 Their expressions are highly inducible by cytokines and lipopolysaccharide (LPS). [17][18][19][20] They are coinduced in inflammatory cells and their products work in concert to cause tissue inflammation and damage. 7 Transcriptional regulation of COX2 and INOS in murine RAW 264.7 ce...

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 exhibits anti-inflammatory and anticancer effects and could be a chemopreventive and chemotherapeutic agent against cancers, but the precise mechanisms involved remain largely unresolved. In this study, we evaluated the mechanism of action of melatonin in human MDA-MB-361 breast cancer cells. Melatonin at pharmacological concentrations (10(-3)  m) significantly suppressed cell proliferation and induced apoptosis in a dose-dependent manner. The observed suppression of proliferation was accompanied by the melatonin-mediated inhibition of COX-2, p300, and NF-κB signaling. Melatonin significantly inhibited COX-2 expression and prostaglandin E(2) (PGE2) production, abrogated p300 histone acetyltransferase activity and p300-mediated NF-κB acetylation, thereby blocking NF-κB binding and p300 recruitment to COX-2 promoter. Pretreatment with a COX-2- or p300-selective inhibitor abrogated the melatonin-induced inhibition of cell proliferation, whereas PGE2 treatment or COX-2 transfection reversed the inhibition by melatonin. Moreover, melatonin markedly inhibited phosphorylation of PI3K, Akt, PRAS40, and GSK-3 proteins, thereby inactivating the PI3K/Akt signaling pathway. Pretreatment with a PI3K- or an Akt-selective inhibitor or an Akt-specific siRNA blocked the melatonin-mediated inhibition of cell proliferation. Conversely, gene delivery of a constitutively active Akt effectively reversed the inhibition by melatonin. Furthermore, melatonin induced Apaf-1 expression, triggered cytochrome C release, and stimulated caspase-3 and caspase-9 activities and cleavage, leading to an activation of the Apaf-1-dependent apoptotic pathway. Pretreatment with an Apaf-1-specific siRNA effectively attenuated the melatonin-induced apoptosis. These results therefore indicate that melatonin inhibits cell proliferation and induces apoptosis in MDA-MB-361 breast cancer cells in vitro by simultaneously suppressing the COX-2/PGE2, p300/NF-κB, and PI3K/Akt/signaling and activating the Apaf-1/caspase-dependent apoptotic pathway.

Coactivators p300 and CREB (cyclic adenosine monophosphate [cAMP]–response element binding protein)–binding protein (CBP) serve as an integrator for gene transcription. Their relative involvement in regulating cyclooxygenase-2 (COX-2) promoter activity had not been characterized. Using fibroblast and macrophage COX-2 transcription as a model, we determined p300 and CBP levels in nuclear extracts and their binding to a COX-2 promoter probe. CBP level was barely detectable and there was little CBP binding. In contrast, p300 was detectable in nucleus and its binding to a COX-2 promoter probe was enhanced by phorbol 12-myristate 13-acetate (PMA), interleukin-1β (IL-1β), or lipopolysaccharide (LPS). Binding of p300/CBP-associated factor (PCAF) was also up-regulated. COX-2 proteins and promoter activities induced by these agonists were augmented by p300 overexpression. Early region 1A (E1A), but not its deletion mutant, abrogated COX-2 expression induced by inflammatory mediators and with or without p300 overexpression. Molecular analysis of p300 revealed the requirement of multiple domains, including histone acetyltransferase (HAT) for COX-2 transactivation. Furthermore, roscovitine, an indirect inhibitor of p300 HAT, and histone deacetylase-1 transfection completely abolished COX-2 promoter activity. We conclude that p300 is the predominant coactivator that is essential for COX-2 transcriptional activation by proinflammatory mediators.

BackgroundGlycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3β in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent.MethodsWe used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3β expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5–8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3β in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3β inhibition on the nuclear factor-κB (NF-κB) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3β activity with overall survival.ResultsOsteosarcoma cells with low levels of inactive p-Ser9-GSK-3β formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3β had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3β resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3β resulted in inhibition of the NF-κB pathway and reduction of NF-κB-mediated transcription. Combination treatments with GSK-3β inhibitors, NF-κB inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3β, and nuclear NF-κB had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3β and NF-κB (109.2 months).ConclusionGSK-3β activity may promote osteosarcoma tumor growth, and therapeutic targeting of the GSK-3β and/or NF-κB pathways may be an effective way to enhance the therapeutic activity of anticancer drugs against osteosarcoma.

It is well established that p300 plays an important role in mediating gene expressions. However, it is less clear how its binding is influenced by physiological stimuli and how its altered binding affects transactivator acetylation and binding. In this study, we determined p300 binding to a core cyclooxygenase-2 (COX-2) promoter region by chromatin immunoprecipitation and streptavidin-agarose pull-down assays in basal and tumor necrosis factor-␣ (TNF␣)-treated human foreskin fibroblasts. We found basal binding of p300, p50/p65 NF-B, cyclic AMP regulatory element-binding protein-2, CCAAT/enhancer-binding protein ␤, and c-Jun. p50/p65 and p300 binding was selectively increased by TNF␣. Immunoprecipitation confirmed direct interaction of p300 with NF-B and the other involved transactivators. p50 acetylation was detected in resting cells and was increased by TNF␣ or lipopolysaccharide. Overexpression of p300 augmented p50 acetylation, which was attenuated by deletion of its histone acetyltransferase domain. Enhanced p50 acetylation correlated with increased p50 binding to COX-2 promoter and transcriptional activation. Co-transfection of E1A with p300 abrogated p50 acetylation and p50 binding. These findings suggest that up-regulation of p300 binding and its acetylation of NF-B occupies a central position in COX-2 promoter activation.

Programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) interaction plays a crucial role in tumor-associated immune escape. Here, we verify that triple-negative breast cancer (TNBC) has higher PD-L1 expression than other subtypes. We then discover that nucleophosmin (NPM1) binds to PD-L1 promoter specifically in TNBC cells and activates PD-L1 transcription, thus inhibiting T cell activity in vitro and in vivo. Furthermore, we demonstrate that PARP1 suppresses PD-L1 transcription through its interaction with the nucleic acid binding domain of NPM1, which is required for the binding of NPM1 at PD-L1 promoter. Consistently, the PARP1 inhibitor olaparib elevates PD-L1 expression in TNBC and exerts a better effect with anti-PD-L1 therapy. Together, our research has revealed NPM1 as a transcription regulator of PD-L1 in TNBC, which could lead to potential therapeutic strategies to enhance the efficacy of cancer immunotherapy.

Background: Immune cells have essential auxiliary functions and influence clinical outcomes in cancer, with high immune infiltration being associated with improved clinical outcomes and better response to treatment in breast cancer (BC). However, studies to date have not fully considered the tumor-infiltrating immune cell (TIIC) landscape in tumors. This study investigated potential biomarkers based on TIICs to improve prognosis and treatment effect in BC. Results: We enrolled 5112 patients for analysis and used cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT), a new computational algorithm, to quantify 22 TIICs in primary BC. From the results of univariate Cox regression, 12 immune cells were determined to be significantly related to the overall survival (OS) of BC patients. Furthermore, least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analyses were applied to construct an immune prognostic model based on six potential biomarkers. By dividing patients into low- and high-risk groups, a significant distinction in OS was found in the training cohort, with 20-year survival rates of 42.6% and 26.3%, respectively. Applying a similar protocol to validation and test cohorts, we found that OS was significantly shorter in the high-risk group than in the low-risk group, regardless of the molecular subtype of BC. Using the immune score model to predict the effect of BC patients to chemotherapy, the survival advantage for the low-risk group was evident among those who received chemotherapy, regardless of the chemotherapy regimen. In evaluating the predictive value of the nomogram, a decision curve showed better predictive accuracy than the standard tumor-node-metastasis (TNM) staging system. Conclusion: The immune cell infiltration-based immune score model can be effectively and efficiently used to predict the prognosis of BC patients as well as the effect of chemotherapy.