Inhibitory Effect Of Nobiletin Research Articles

Anti-Inflammatory Activity of the Combination of Nobiletin and Docosahexaenoic Acid in Lipopolysaccharide-Stimulated RAW 264.7 Cells: A Potential Synergistic Anti-Inflammatory Effect.

This study aimed to investigate a synergistic anti-inflammatory effect of a citrus flavonoid nobiletin and docosahexaenoic acid (DHA), one of n-3 long-chain polyunsaturated fatty acids, in combination. Simultaneous treatment with nobiletin and DHA synergistically inhibited nitric oxide production (combination index < 0.9) by mouse macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) without cytotoxicity. On the other hand, the inhibitory effect of nobiletin and DHA in combination on proinflammatory cytokine production was not synergistic. Neither nobiletin nor DHA affected the phagocytotic activity of RAW 264.7 cells stimulated with LPS. Immunoblot analysis revealed that the inhibition potency of DHA on the phosphorylation of ERK and p38 and nuclear translocation of NF-κB is markedly enhanced by simultaneously treating with nobiletin, which may lead to the synergistic anti-inflammatory effect. Overall, our findings show the potential of the synergistic anti-inflammatory effect of nobiletin and DHA in combination.

Nobiletin inhibits de novo FA synthesis to alleviate gastric cancer progression by regulating endoplasmic reticulum stress

BackgroundGastric cancer (GC) is a common malignant tumor with limited treatment options. The natural flavonoid nobiletin (NOB) is a beneficial antioxidant that possesses anticancer activity. However, the mechanisms by which NOB inhibits GC progression remain unclear. MethodsA CCK-8 assay was performed to determine cytotoxicity. Cell cycle and apoptosis analyses were performed by flow cytometry. RNA-seq was performed to detect differential gene expression after NOB treatment. RT‒qPCR, Western blot and immunofluorescence staining were used to examine the underlying mechanisms of NOB in GC. Xenograft tumor models were constructed to verify the effect of NOB and its specific biological mechanism in GC. ResultsNOB inhibited cell proliferation, caused cell cycle arrest and induced apoptosis in GC cells. KEGG classification identified that the inhibitory effect of NOB on GC cells mainly involved the lipid metabolism pathway. We further showed that NOB reduced de novo fatty acid (FA) synthesis, as evidenced by the decreased levels of neutral lipids and the expression levels of ACLY, ACACA and FASN, and ACLY abrogated the effect of NOB on lipid deposits in GC cells. In addition, we also found that NOB triggered endoplasmic reticulum (ER) stress by activating the IRE-1α/GRP78/CHOP axis, but overexpression of ACLY reversed ER stress. Mechanistically, inhibiting ACLY expression with NOB significantly reduced neutral lipid accumulation, thereby inducing apoptosis by activating IRE-1α-mediated ER stress and inhibiting GC cell progression. Finally, in vivo results also demonstrated that NOB inhibited tumor growth by decreasing de novo FA synthesis. ConclusionNOB could inhibit the expression of ACLY to activate IRE-1α-induced ER stress, which ultimately led to GC cell apoptosis. Our results provide novel insight into the use of de novo FA synthesis for GC treatment and are the first to reveal that NOB inhibits GC progression by ACLY-dependent ER stress.

Inhibitory Effects of Nobiletin on Voltage-Gated Na+ Channel in Rat Ventricular Myocytes Based on Electrophysiological Analysis and Molecular Docking Method.

Nobiletin (NOB) has attracted much attention owing to its outstanding bioactivities. This study aimed to investigate its anti-arrhythmic effect through electrophysiological and molecular docking studies. We assessed the anti-arrhythmic effects of NOB using aconitine-induced ventricular arrhythmia in a rat model and the electrophysiological effects of NOB on rat cardiomyocytes utilizing whole-cell patch-clamp techniques. Moreover, we investigated the binding characters of NOB with rNav1.5, rNav1.5/QQQ, and hNaV1.5 via docking analysis, comparing them with amiodarone and aconitine. NOB pretreatment delayed susceptibility to ventricular premature and ventricular tachycardia and decreased the incidence of fatal ventricular fibrillation. Whole-cell patch-clamp assays demonstrated that the peak current density of the voltage-gated Na+ channel current was reversibly reduced by NOB in a concentration-dependent manner. The steady-state activation and recovery curves were shifted in the positive direction along the voltage axis, and the steady-state inactivation curve was shifted in the negative direction along the voltage axis, as shown by gating kinetics. The molecular docking study showed NOB formed a π-π stacking interaction with rNav1.5 and rNav1.5/QQQ upon Phe-1762, which is the homolog to Phe-1760 in hNaV1.5 and plays an important role in antiarrhythmic action This study reveals that NOB may act as a class I sodium channel anti-arrhythmia agent.

Nobiletin Inhibits Non-Small-Cell Lung Cancer by Inactivating WNT/β-Catenin Signaling through Downregulating miR-15-5p.

Background Nobiletin is a natural compound with anticancer activity; however, the mechanism is not clear. Methods The inhibitory effect of nobiletin on non-small-cell lung cancer (NSCLC) cells was examined using soft agar, Transwell, and apoptosis analyses. Cancer stemness was measured by sphere assay. Genes and miRNAs regulated by nobiletin were identified by whole-genome sequencing. Protein levels were detected by western blot and immunofluorescence assays. Results Nobiletin significantly inhibited NSCLC cell colony formation and sphere formation and induced apoptosis. Nobiletin upregulated negative regulators of WNT/β-catenin signaling, including NKD1, AXIN2, and WIF1, while it inhibited the expression of β-catenin and its downstream genes, including c-Myc, c-Jun, and cyclin D1. Furthermore, we identified that GN inhibits miR-15-5p expression in NSCLC cells and that NKD1, AXIN2, and WIF1 are the target genes of miR-15-5p. Conclusions Nobiletin has a strong inhibitory effect on NSCLC, and nobiletin plays an anticancer role by inhibiting miR-15-5p/β-catenin signaling in NSCLC.

Anti-inflammatory effects of nobiletin on TLR4/TRIF/IRF3 and TLR9/IRF7 signaling pathways in prostate cancer cells

Background: Toll-like receptors (TLRs) are often expressed in natural immune cells as well as in tumor cells. TLR4 exhibits both tumor promoting and tumor-suppressing roles and higher TLR9 expression is an important marker of poor prognosis in prostate cancer (PCa). Nobiletin (NOB) is an O-methylated flavonoid and NOB has been proven to have anti-cancer effect in PCa cells. However, there is no study in the literature investigating the potential anti-inflammatory effects of NOB on the TLR signaling pathways in cancer. Therefore, we aimed to explore the potential anti-inflammatory effects of NOB on the TLR4/TRIF/IRF3 and TLR9/IRF7 signaling pathways in different types of PCa cell lines, for the first time.Material and methods: In the current study, the cytotoxic effect of NOB PC-3 (hormone-independent and metastatic) and LNCaP cells (hormone-dependent) was evaluated by WST-1 assay. Furthermore, the inhibitory effects of NOB on TLR4/TRIF/IRF3 and TLR9/IRF7signaling pathway were determined by RT-PCR, western blotting and ELISA analysis.Results: NOB demonstrated an inhibitory effect on PCa cell growth and LNCaP cells were more sensitive to NOB than PC-3 cells due to androjen receptor status. Furthermore, NOB alone could suppress TLR4/TRIF/IRF3 and TLR9/IRF7 signaling pathways through the downregulation of their associated pathways (mRNA and related protein levels) and the release of IFN-α and IFN-β compared to LPS or CpG-ODN stimulated PCa cells.Conclusions: NOB potentially inhibited TLR4 and TL9-dependent signaling pathway in PCa cells. However, the efficacy of NOB was different in PCa cells due to the hormone status and aggressive features.

Inhibitory effects of nobiletin and its major metabolites on lung tumorigenesis.

Nobiletin (NBT), a citrus flavonoid, has been associated with various health benefits. Herein, we investigated the chemopreventive actions of NBT and its metabolites in a pulmonary carcinogenesis mouse model and human lung cancer cells. In 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-treated mice, the oral administration of NBT significantly suppressed lung tumorigenesis as evidenced by reduced tumor volume compared to the control mice. NBT also greatly attenuated cell proliferation in the lung of NNK-treated mice. Our previous study has identified three major metabolites of NBT, namely, 3'-demethylnobiletin (M1), 4'-demethylnobiletin (M2), and 3',4'-didemethylnobiletin (M3). In this study, we further determined the inhibitory effects of NBT and its metabolites on human non-small cell lung cancer (NSCLC) cells and the underlying mechanisms of action. Interestingly, we found that M2 and M3 exerted much stronger growth inhibition on both H460 and H1299 cells, compared to their parent compound NBT. Flow cytometry and western blotting analysis revealed that M2 and M3 caused significant cell cycle arrest and cellular apoptosis and profoundly modulated multiple proteins associated with cell proliferation and cell death, including p21, cyclin B1, CDK1, cyclin D1, CDK6, CDK4, Bax, cleaved caspase-1, and cleaved PARP. Overall, our results demonstrated that the oral administration of NBT significantly inhibited lung carcinogenesis in mice, and these chemopreventive effects could be attributed to its metabolites that showed potent anti-cancer effects.

Inhibitory Effects of Nobiletin on Colitis were Associated with Altered Gut Microbiota

Understanding how dietary compounds such as nobiletin alter the colonic environment is critical in studying their roles in modulating gut health and gut associated diseases. In this study, we determined the effects of dietary nobiletin treatment on dextran sodium sulfate (DSS)‐induced colitis as well as the composition of gut microbiota in mice. The results revealed that nobiletin treatment significantly decreased the severity of colonic inflammation in DSS‐treated mice, evidenced by the reduced production of pro‐inflammatory cytokines (i.e., GM‐CSF, INF‐γ, IL‐1β, IL‐2, IL‐6, KC/GRO, and TNF‐α) in the colonic mucosa, increased colon length, and decreased disease activity index and histologic score of inflammation (p&lt;0.05) by the nobiletin treatment. Results from next generation sequencing of the fecal microbiota showed that DSS treatment significantly altered the microbial structure of fecal microbiota in mice, and dietary nobiletin treatment tend to reverse these alterations. It was also demonstrated that DSS treatment decreased the abundance of Bifidobacterium and Lactobacillus compared to the negative control (healthy) mice. Nobiletin treatment partially reversed the DSS‐induced decrease in these two genera in DSS treated mice. It is noteworthy that nobiletin had no effect on Bifidobacterium, but increased the abundance of Lactobacillus in the healthy mice (non‐DSS treated). DSS treatment significantly increased the abundance of Sutterella and Bilophila, while nobiletin effectively reversed DSS‐induced increases in these two genera. Overall, our results for the first time suggested that modification of gut microbiota by dietary nobiletin might contribute to its inhibitory effects against the development of colitis in mice.

Nobiletin inhibits human osteosarcoma cells metastasis by blocking ERK and JNK-mediated MMPs expression.

Nobiletin, a polymethoxyflavone, has a few pharmacological activities, including anti-inflammation and anti-cancer effects. However, its effect on human osteosarcoma progression remains uninvestigated. Therefore, we examined the effectiveness of nobiletin against cellular metastasis of human osteosarcoma and the underlying mechanisms. Nobiletin, up to 100 μM without cytotoxicity, significantly decreased motility, migration and invasion as well as enzymatic activities, protein levels and mRNA expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in U2OS and HOS cells. In addition to inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the inhibitory effect of nobiletin on the DNA-binding activity of the transcription factor nuclear factor-kappa B (NF-κB), cAMP response element-binding protein (CREB), and specificity protein 1 (SP-1) in U2OS and HOS cells. Co-treatment with ERK and JNK inhibitors and nobiletin further reduced U2OS cells migration and invasion. These results indicated that nobiletin inhibits human osteosarcoma U2OS and HOS cells motility, migration and invasion by down-regulating MMP-2 and MMP-9 expressions via ERK and JNK pathways and through the inactivation of downstream NF-κB, CREB, and SP-1. Nobiletin has the potential to serve as an anti-metastatic agent for treating osteosarcoma.

Anti-neuroinflammatory Activity of Nobiletin on Suppression of Microglial Activation

A growing body of evidence suggests that nobiletin (5,6,7,8,3',4'-hexamethoxy flavone) from the peel of citrus fruits, enhances the damaged cognitive function in disease animal models. However, the neuroprotective mechanism has not been clearly elucidated. Since nobiletin has shown anti-inflammatory effects in several tissues, we investigated whether nobiletin suppresses excessive microglial activation implicated in neurotoxicity in lipopolysaccharide (LPS)-stimulated BV-2 microglia cell culture models. Release of nitric oxide (NO), the major inflammatory mediator in microglia, was markedly suppressed in a dose-dependent manner following nobiletin treatment (1-50 µM) in LPS-stimulated BV-2 microglia cells. The inhibitory effect of nobiletin was similar to that of minocycline, a well-known microglial inactivator. Nobiletin significantly inhibited the release of the pro-inflammatory cytokine tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β). LPS-induced phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) were also significantly inhibited by nobiletin treatment. In addition, nobiletin markedly inhibited the LPS-induced pro-inflammatory transcription factor nuclear factor κB (NF-κB) signaling pathway by suppressing nuclear NF-κB translocation from the cytoplasm and subsequent expression of NF-κB in the nucleus. Taken together, these results may contribute to further exploration of the therapeutic potential and molecular mechanism of nobiletin in relation to neuroinflammation and neurodegenerative diseases.

Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts

The inhibitory effects of nobiletin and hesperidin from citrus peel crude extracts on tyrosinase diphenolase activity are evaluated. IC50 of nobiletin and hesperidin is 1.49 mM and 16.08 mM, respectively and their inhibition mechanism is competitive type with Ki = 2.82 mM and noncompetitive with Ki = 9.16 mM, respectively. Crude extracts from citrus peel (C. unshiu Marc.) were extracted with 95% ethanol and fractionated by petroleum ether (PCPE). The ethanol phase (ECPE) was further desorbed from macroporous adsorption resin (FGRE). Their IC50 values were 8.09 mg/mL, 7.53 mg/mL and 4.80 mg/mL, respectively. Their inhibition on melanogenesis in B16 mouse melanoma cells was also evaluated. FGRE showed a significant inhibition (42.5% at 31.25 μg/mL, p < 0.01) while hesperidin showed almost no inhibition. Nobiletin and PCPE give efficacious antiproliferation effects on B16 mouse melanoma cell with IC50 values 88.6 μM and 62.96 μg/mL, respectively, by the MTT test. Hesperidin and other crude extracts showed very low cytotoxity to the B16 cell.

Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts

The inhibitory effects of nobiletin and hesperidin from citrus peel crude extracts on tyrosinase diphenolase activity have been evaluated. IC50 of nobiletin and hesperidin were 1.49 mM and 16.08 mM, respectively and their inhibition mechanisms are competitive type with inhibition constant (Ki) 2.82 mM and noncompetitive type with Ki 9.16 mM, respectively. Crude extracts from citrus peel (C. unshiu Marc.) were extracted with 95% ethanol and fractionated by petroleum ether (PCPE). The ethanol phase (ECPE) was further desorbed from macroporous adsorption resin (FGRE). Their IC50 values were 8.09 mg/mL, 7.53 mg/mL and 4.80 mg/mL, respectively. Their inhibition of melanogenesis in B16 mouse melanoma cells was also evaluated. FGRE showed a significant inhibition (42.48% at 31.25 μg/mL, p < 0.01) while hesperidin showed almost no inhibition. Nobiletin and PCPE gave efficacious antiproliferation effects on the B16 mouse melanoma cell with IC50 values 88.6 μM and 62.96 μg/mL, respectively, through the MTT test. Hesperidin and other crude extracts showed very low cytotoxity to the B16 cell.