Gamma irradiation of aloe-emodin induced structural modification and apoptosis through a ROS- and caspase-dependent mitochondrial pathway in stomach tumor cells.

PURPOSE: The changes in molecular structure and the physiological properties of a gamma-irradiated aloe-emodin were examined. MATERIALS AND METHODS: Aloe-emodin was gamma-irradiated at doses ranging from 0 to 150 kGy, and the molecular structure was then analyzed using high-performance liquid chromatography (HPLC). AGS cells were cultured in RPMI medium and treated gamma irradiated aloe-emodin. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis efficiency was investigated by cell cycle arrest, cell morphology, and signaling pathway. The structure of new radiolytic peak was identified by the hydrogen-nuclear magnetic resonance (1H NMR). RESULTS: HPLC results showed that gamma irradiation induced new radiolytic peaks that were distinguishable from the aloe-emodin standard, and the area of new peaks was increased as the radiation dose increased. Gamma-irradiated aloe-emodin treatment significantly increased the cytotoxicity in AGS tumor cells. We also found that 150 kGy aloe-emodin increased the expression of Bax, cytosolic cytochrome c, PARP cleavage, and the activation of caspases-8, -9, -3, Bid, and Bcl-2. Treatment of 150 kGy aloe-emodin induced ROS production, DNA fragmentation, alterations of cell morphology, and the migration in AGS cells. Gamma-irradiated aloe-emodin induced an increase of sub-G1 phase and depolarization of mitochondrial membrane potential in AGS cells. We also confirmed that fractionated AEF1 (new radiolytic peak) induce the cell death, migration, an increase of sub-G1 phase and cytochrome c in a ROS-dependent manner. CONCLUSIONS: The radiolysis product (AEF1) of aloe-emodin transformed by gamma-irradiation strongly induced apoptotic cell death in AGS cells, indicating AEF1 is a potential candidate drug for use in anti-cancer drug.

Gamma-Irradiated Luteolin Inhibits 3-Isobutyl-1-Methylxanthine-Induced Melanogenesis Through the Regulation of CREB/MITF, PI3K/Akt, and ERK Pathways in B16BL6 Melanoma Cells.

Luteolin was gamma irradiated at doses of 0, 15, 30, 50, 70, and 100 kGy. We observed that the luteolin peak decreased simultaneously with the appearance of new radiolytic peaks, using high-performance liquid chromatography (HPLC). The highest new radiolytic peak (GLM) of radiolytic product in gamma-irradiated luteolin was observed at a dose of 70 kGy, and the GLM was identified by nuclear magnetic resonance and high-performance-liquid-chromatography-quadrupole-time-of-flight (HPLC-Q-TOF) mass spectrometry. We examined whether 70 kGy gamma-irradiated luteolin has more effective anti-melanogenic effects than intact luteolin. Seventy kilograys of gamma-irradiated luteolin inhibited melanin synthesis and intracellular tyrosinase activity without cytotoxicity, whereas the intact luteolin-treated group did not show anti-melanogenic activity in 3-isobutyl-1-methylxanthine-stimulated B16BL6 melanoma cells. The expression of melanogenic enzymes, such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2, was decreased by 70 kGy gamma-irradiated luteolin treatment, owing to the suppression of microphthalamia-associated transcription factor and 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein. In addition, gamma-irradiated luteolin decreased the phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt and extracellular regulated kinase (ERK). The anti-melanogenic effects of 70 kGy gamma-irradiated luteolin were attenuated by the treatment of two specific inhibitors (PD98059 and LY294002), and these results indicate that the anti-melanogenic effects were mediated by ERK and PI3K signaling pathways. Therefore, our findings suggest that gamma-irradiated luteolin can be a potential cosmeceutical agent for skin whitening.

Gamma irradiation enhanced Tollip-mediated anti-inflammatory action through structural modification of quercetin in lipopolysaccharide-stimulated macrophages.

The changes in molecular structure and anti-inflammatory action of a gamma-irradiated quercetin were examined. Quercetin was gamma-irradiated at doses of 0, 15, 30, 50, 100 and 150kGy, which induced new radiolytic peaks (the highest radiolytic peak at a dose of 30kGy). Treatment of intact- and gamma-irradiated quercetin did not induce a significant cellular toxicity of macrophages at concentrations ranging from 12.5 to 50µM. Treatment of LPS-stimulated macrophages with gamma-irradiated quercetin (30kGy) showed a higher inhibitory action than intact-quercetin groups in the excessive expression of inducible nitric oxide synthases-mediated nitric oxide, prostaglandin E2, pro-inflammatory cytokines level, such as tumor necrosis factor-α, interleukin-6 and interleukin-1ß, reactive oxygen species, as well as cell surface molecules (CD80, CD86, and MHC class I/II). The inhibition of LPS-stimulated pro-inflammatory mediators was mediated through a suppression of mitogen-activated protein kinases and nuclear factor-κB pathways. In addition, gamma-irradiated quercetin (30kGy) markedly elevated the expression of the Toll-interacting protein compared to intact-quercetin. The inhibitory action of intact- and gamma-irradiated quercetin on the production of IL-6 and TNF-α was not observed in the down-regulation of Tollip. Therefore, these findings represent new insights into the understanding of the changes in molecular structure and the physiological properties of natural products through the application of radiation technology.

Gamma-irradiated resveratrol negatively regulates LPS-induced MAPK and NF-κB signaling through TLR4 in macrophages.

Resveratrol was irradiated at various doses of 15, 30, 50, and 70kGy for the development of physiological functionalities through modification of the structural properties. Gamma irradiation induced a decrease in the resveratrol peak, and the appearance of several new peaks by gamma irradiation was gradually increased up to 70kGy. Gamma-irradiated resveratrol did not exert cytotoxicity to macrophages in dose ranges from 15 to 70kGy; therefore, 70kGy gamma-irradiated resveratrol was used as the maximum dose throughout subsequent experiments. Treatment of LPS-stimulated macrophages with 70kGy gamma-irradiated resveratrol resulted in a dose-dependent decrease in iNOS-mediated NO, PGE2, and pro-inflammatory cytokine level, such as TNF-α, IL-6 and IL-1ß. 70kGy gamma-irradiated resveratrol significantly inhibited cyclooxygenase-2 levels, as well as the expression of cell surface molecules, such as CD80 and CD86, in LPS-induced macrophages. Furthermore, the inhibitory action of these pro-inflammatory mediators occurred through an inhibition of MAPKs (ERK1/2, p38 and JNK) and NF-κB signaling pathways based on a toll-like receptor 4 in macrophages, which may be closely mediated with the radiolysis products of resveratrol transformed by gamma-irradiation. From these findings, it seems likely that gamma irradiation can be an effective tool for a reduction of the toxicity and play a potent role in the treatment of inflammatory disease.

Anti-inflammatory effect of gamma-irradiated genistein through inhibition of NF-κB and MAPK signaling pathway in lipopolysaccharide-induced macrophages.

Genistein was irradiated with γ-irradiation at doses of 0, 10, 30, 50, 100, and 150 kGy. We observed that the decrease in the genistein peak after gamma irradiation was concomitant with the appearance of several new peaks. 150 kGy gamma-irradiated genistein did not exert cytotoxicity in macrophages, and inhibited inducible nitric oxide synthase-mediated nitric oxide production and pro-inflammatory cytokines level, such as tumor necrosis factor-α, interleukin-6 and interleukin-1ß, in lipopolysaccharide (LPS)-induced macrophages. The treatment of LPS-stimulated macrophages with 150 kGy gamma-irradiated genistein resulted in a significant decrease in cyclooxygenase-2 levels, as well as the expression of cell surface molecules, such as CD80 and CD86. Furthermore, we also found that the anti-inflammatory action of 150 kGy gamma-irradiated genistein occurred through an inhibition of mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, p38 and c-Jun N-terminal kinase) and nuclear factor-κB signaling pathways based on a toll-like receptor 4 in macrophages, which may be speculated that several radiolysis products of genistein transformed by gamma-irradiation induce the inhibition of pro-inflammatory mediators. From these findings, it seems likely that gamma-irradiated genistein could play a potent role in the treatment of inflammatory disease as a value-added product in the medical industry.

Procyanidin C1 causes vasorelaxation through activation of the endothelial NO/cGMP pathway in thoracic aortic rings.

The aim of this study was to clarify the efficacy of procyanidin C1 (Pro C1) for modulating vascular tone. Pro C1 induced a potent vasorelaxant effect on phenylephrine-constricted endothelium-intact thoracic aortic rings, but had no effect on denuded thoracic aortic rings. Moreover, Pro C1 caused a significant increase in nitric oxide (NO) production in endothelial cells. Pro C1-induced vasorelaxation and Pro C1-induced NO production were significantly decreased in the presence of a nonspecific potassium channel blocker (tetraethylammonium chloride [TEA]), an endothelial NO synthase inhibitor (N(G)-monomethyl-L-arginine [L-NMMA]), and a store-operated calcium entry inhibitor (2-aminoethyl diphenylborinate [2-APB]). Pro C1-induced vasorelaxation was also completely abolished by an inhibitor of soluble guanyl cyclase, which suggests that the Pro C1 effects observed involved cyclic guanosine monophosphate (cGMP) production. Interestingly, Pro C1 significantly enhanced basal cGMP levels. Taken together, these results indicate that Pro C1-induced vasorelaxation is associated with the activation of the calcium-dependent NO/cGMP pathway, involving potassium channel activation. Thus, Pro C1 may represent a novel and potentially therapeutically relevant compound for the treatment of cardiovascular diseases.

Procyanidin dimer B2-mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages.

Polyphenolic compounds have been found to possess a wide range of physiological activities that may contribute to their beneficial effects against inflammation-related diseases; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. In this study, we investigated the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by procyanidin dimer B2 (Pro B2) in macrophages. Pro B2 markedly elevated the expression of the interleukin (IL)-1 receptor-associated kinase (IRAK)-M protein, a negative regulator of TLR signaling. Lipopolysaccharide (LPS)-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1ß, IL-6, and IL-12p70) were inhibited by Pro B2, and this action was prevented by IRAK-M silencing. In addition, Pro B2-treated macrophages inhibited LPS-induced activation of mitogen-activated protein kinases such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase and the translocation of nuclear factor κB and p65 through IRAK-M. We also found that Pro B2-treated macrophages inactivated naïve T cells by inhibiting LPS-induced interferon-γ and IL-2 secretion through IRAK-M. These novel findings provide new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and the immune-pharmacological role of Pro B2 in the immune response against the development and progression of many chronic diseases.

The procyanidin trimer C1 induces macrophage activation via NF-κB and MAPK pathways, leading to Th1 polarization in murine splenocytes.

Numerous studies have shown various relationships between foods with a high nutritional value and a robust immune response, particularly studies that have focused on host protection and cytokine networks. This study aimed to clarify the role played by the procyanidin trimer C1 in innate and adaptive immunity. Procyanidin C1 did not exert cytotoxicity at concentrations ranging from 7.8 to 62.5 µg/ml in macrophage cells; therefore, concentration of 62.5 µg/ml was used as the maximum dose of procyanidin C1 throughout subsequent experiments. Procyanidin C1 enhanced inducible nitric oxide synthase-mediated nitric oxide production in a concentration-dependent manner. In addition, procyanidin C1 functionally induced macrophage activation by augmenting the expression of cell surface molecules (CD80, CD86, and MHC II) and proinflammatory cytokine production (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6) via activation of mitogen-activated protein kinase (MAPK), e.g., p38, ERK, and JNK and nuclear factor (NF)-κB signaling pathways. Interestingly, procyanidin C1 effectively polarized T helper type 1 (Th1) by secreting Th1-mediated cytokines (interferon-γ, IL-12p70, and IL-2) and inducing splenocyte proliferation, indicating that procyanidin C1 contributes to Th1 polarization of the immune response. Accordingly, these findings confirms that the procyanidin C1 induces macrophage activation via NF-κB and MAPK pathways, leading to Th1 polarization in murine splenocytes, which suggests that procyanidin C1 regulates innate and adaptive immunity by macrophage activation and Th1 polarization.

Quercetin negatively regulates TLR4 signaling induced by lipopolysaccharide through Tollip expression.

Polyphenolic compounds have been regarded as one of the most promising dietary agents for the prevention and treatment of inflammation-related chronic diseases; however, the anti-inflammatory activities of flavonoids, such as quercetin, are not completely characterized, and many features remain to be elucidated. In this study, we showed the molecular basis for the downregulation of TLR4 signal transduction by quercetin. Quercetin markedly elevated the expression of the Toll-interacting protein, a negative regulator of TLR signaling. Lipopolysaccharide-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1ß, IL-6, and IL-12p70) were inhibited by quercetin, and this action was prevented by Toll-interacting protein silencing. In addition, quercetin-treated macrophages inhibited lipopolysaccharide-induced activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase, and the translocation of nuclear factor-κB and p65 through Toll-interacting protein. Treatment with quercetin resulted in a significant decrease in prostaglandin E2 and cyclooxygenase-2 levels as well as inducible nitric oxide synthase-mediated nitric oxide production induced by lipopolysaccharide. Taken together, these findings represent new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and effective therapeutic intervention for the treatment of inflammatory disease.