Stigmasterol Exerts an Anti-Melanoma Property through Down-Regulation of Reactive Oxygen Species and Programmed Cell Death Ligand 1 in Melanoma Cells.

Cancer immunotherapy as a promising anti-cancer strategy has been widely studied in recent years. Stigmasterol (STIG), a phytosterol, is known to have various pharmacological effects, including anti-inflammatory effects. However, the pharmacological role of STIG on melanoma immunotherapy has not been investigated. The present study demonstrates the anti-melanoma potency of STIG through the regulation of PD-L1 levels. The results reveal that STIG reduces reactive oxygen species (ROS) levels induced by hydrogen peroxide and increases glutathione levels decreased by α-MSH in B16F10 cells. Moreover, STIG significantly decreases melanin content and tyrosinase activities elevated by α-MSH. It also suppresses nitric oxide production induced by α-MSH. Additionally, STIG induces apoptosis with the up-regulation of PARP activation. STIG inhibits IFN-γ-induced PD-L1 expression and STAT1 phosphorylation levels. STIG also reverses the up-regulation of PD-L1 and phosphorylated STAT1 levels augmented by cisplatin, and STIG enhances CD8(+) T-cell-mediated cell death against B16F10 cells. These findings represent the first evidence of pro-apoptotic activity of STIG on melanoma cells through the down-regulation of ROS and PD-L1 pathways. Therefore, STIG may be an effective candidate for melanoma immunotherapy.

Maltol has anti-cancer effects via modulating PD-L1 signaling pathway in B16F10 cells.

Introduction: Among skin cancers, melanoma has a high mortality rate. Recent advances in immunotherapy, particularly through immune checkpoint modulation, have improved the clinical treatment of melanoma. Maltol has various bioactivities, including anti-oxidant and anti-inflammatory properties, but the anti-melanoma property of maltol remains underexplored. The aim of this work is to explore the anti-melanoma potential of maltol through regulating immune checkpoints. Methods: The immune checkpoint PD-L1 was analyzed using qPCR, immunoblots, and immunofluorescence. Melanoma sensitivity towards T cells was investigated via cytotoxicity, cell viability, and IL-2 assays employing CTLL-2 cells. Results: Maltol was found to reduce melanin contents, tyrosinase activity, and expression levels of tyrosinase and tyrosinase-related protein 1. Additionally, maltol suppressed the proliferative capacity of B16F10 and induced cell cycle arrest. Maltol increased apoptotic rates by elevating cleaved caspase-3 and PARP. The co-treatment with maltol and cisplatin revealed a synergistic effect on inhibiting growth and promoting apoptosis. Maltol suppressed IFN-γ-induced PD-L1 and cisplatin-upregulated PD-L1 by attenuating STAT1 phosphorylation, thereby enhancing cisplatin's cytotoxicity against B16F10. Maltol augmented sensitivity to CTLL-2 cell-regulated melanoma destruction, leading to an increase in IL-2 production. Discussion: These findings demonstrate that maltol restricts melanoma growth through the downregulation of PD-L1 and elicits T cell-mediated anti-cancer responses, overcoming PD-L1-mediated immunotherapy resistance of cisplatin. Therefore, maltol can be considered as an effective therapeutic agent against melanoma.

The Mixture of Natural Products SH003 Exerts Anti-Melanoma Effects through the Modulation of PD-L1 in B16F10 Cells.

Melanoma is the most invasive and lethal skin cancer. Recently, PD-1/PD-L1 pathway modulation has been applied to cancer therapy due to its remarkable clinical efficacy. SH003, a mixture of natural products derived from Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii, and formononetin (FMN), an active constituent of SH003, exhibit anti-cancer and anti-oxidant properties. However, few studies have reported on the anti-melanoma activities of SH003 and FMN. This work aimed to elucidate the anti-melanoma effects of SH003 and FMN through the PD-1/PD-L1 pathway, using B16F10 cells and CTLL-2 cells. Results showed that SH003 and FMN reduced melanin content and tyrosinase activity induced by α-MSH. Moreover, SH003 and FMN suppressed B16F10 growth and arrested cells at the G2/M phase. SH003 and FMN also led to cell apoptosis with increases in PARP and caspase-3 activation. The pro-apoptotic effects were further enhanced when combined with cisplatin. In addition, SH003 and FMN reversed the increased PD-L1 and STAT1 phosphorylation levels induced by cisplatin in the presence of IFN-γ. SH003 and FMN also enhanced the cytotoxicity of CTLL-2 cells against B16F10 cells. Therefore, the mixture of natural products SH003 demonstrates therapeutic potential in cancer treatment by exerting anti-melanoma effects through the PD-1/PD-L1 pathway.

The Protective Effect of a Functional Food Consisting of Astragalus membranaceus, Trichosanthes kirilowii, and Angelica gigas or Its Active Component Formononetin against Inflammatory Skin Disorders through Suppression of TSLP via MDM2/HIF1α Signaling Pathways.

An herbal mixture (SH003) of Astragalus membranaceus, Trichosanthes kirilowii, and Angelica gigas exhibits therapeutic effects on carcinomas and immunosuppression. However, the role of JRP-SNF102, which is an advanced mixture of SH003, in regulating inflammatory responses is unexplored. We aim to substantiate the therapeutic potential of JRP-SNF102 and its active component, formononetin (FMN), as a functional food that moderates inflammatory responses. The inhibitory effects of JRP-SNF102 or FMN on thymic stromal lymphopoietin (TSLP) levels were evaluated in phorbol 12-myristate 13-acetate (PMA) plus A23187-activated human mast cell line-1 (HMC-1) cells and a mouse model of PMA-induced ear edema. The JRP-SNF102 or FMN inhibited the secretion and mRNA expression of TSLP and vascular endothelial growth factor (VEGF) in the activated HMC-1 cells. The expression levels of murine double minute 2 (MDM2), hypoxia-inducible factor 1α (HIF1α), and NF-κB were also suppressed by JRP-SNF102 or FMN in the activated HMC-1 cells. The JRP-SNF102 or FMN inhibited TSLP and VEGF levels, attenuating redness and ear thickness in mice with acute ear edema; JRP-SNF102 or FMN reduced the expression levels of MDM2, HIF1α, and NF-κB in the ear tissues. These findings suggest the potential for JRP-SNF102 as a functional food in the treatment of inflammatory skin disorders through suppression of TSLP and VEGF.

TSLP up-regulates inflammatory responses through induction of autophagy in T cells.

Thymic stromal lymphopoietin (TSLP), a type I cytokine belonging to the IL-2 cytokine family, promotes Th2-mediated inflammatory responses. The aim of this study is to investigate whether TSLP increases inflammatory responses via induction of autophagy using a murine T cell lymphoma cell line, EL4 cells, and lipopolysaccharide (LPS)-injected mice. TSLP increased expression levels of autophagy-related factors, such as Beclin-1, LC3-II, p62, Atg5, and lysosome associated membrane protein 1/2, whereas these factors increased by TSLP disappeared by neutralization of TSLP in EL4 cells. TSLP activated JAK1/JAK2/STAT5/JNK/PI3K, while the blockade of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways reduced the expression levels of Beclin-1, LC3-II, and p62 in TSLP-stimulated EL4 cells. In addition, TSLP simultaneously increased levels of inflammatory cytokines via induction of autophagy by activation of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways. In an LPS-induced acute liver injury (ALI) mouse model, exogenous TSLP increased expression levels of Beclin-1 and LC3-II, whereas functional deficiency of TSLP by TSLP siRNA resulted in lower expression of Beclin-1, LC3-II, and inflammatory cytokines, impairing their ability to form autophagosomes in ALI mice. Thus, our findings show a new role of TSLP between autophagy and inflammatory responses. In conclusion, regulating TSLP-induced autophagy may be a potential therapeutic strategy for inflammatory responses.

The immune-enhancing effects of a mixture of Astragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes Kirilowii (Maxim.) or its active constituent nodakenin.

ETHNOPHARMACOLOGICAL RELEVANCE: A mixture (SH003) of Astragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes Kirilowii (Maxim.) has beneficial effects against several carcinomas. There have been few reports on an immune-enhancing activity of SH003 and its active constituent nodakenin. AIM OF THE STUDY: This study aimed at identifying the immune-enhancing effect of SH003 and nodakenin. MATERIALS AND METHODS: The immune-enhancing effect was evaluated using RAW264.7 macrophages, mouse primary splenocytes, and a cyclophosphamide (CP)-induced immunosuppression murine model. RESULTS: The results show that SH003 or nodakenin stimulated the production levels of granulocyte colony-stimulating factor, IL-12, IL-2, IL-6, TNF-α, and nitric oxide (NO) and the expression levels of iNOS in RAW264.7 macrophages. SH003 or nodakenin also enhanced NF-κB p65 activation in RAW264.7 macrophages. SH003 or nodakenin stimulated the production levels of IFN-γ, IL-12, IL-2, TNF-α, and NO and the expression levels of iNOS in splenocytes. SH003 or nodakenin increased the splenic lymphocyte proliferation and splenic NK cell activity. In addition, SH003 or nodakenin increased the levels of IFN-γ, IL-12, IL-2, IL-6, and TNF-α in the serum and spleen of CP-treated mice, alleviating CP-induced immunosuppression. CONCLUSION: Taken together, the results of this study show that SH003 improved immunosuppression through the activation of macrophages, splenocytes, and NK cells. These findings suggest that SH003 could be applied as a potential immunostimulatory agent for a variety of diseases caused or exacerbated by immunodeficiency.

The immune-enhancing effect of anthocyanin-fucoidan nanocomplex in RAW264.7 macrophages and cyclophosphamide-induced immunosuppressed mice.

Aronia, a healthy fruit well known as black chokeberry, has health-promoting effects on hypertension, oxidative stress, and diabetes. Despite many reports of bioactivities of aronia, there is little scientific research on the potential for immune-enhancement. So, anthocyanin-fucoidan nanocomplex (AFNC, a nanocomplex of aronia extract and fucoidan) has been developed to improve immune-enhancement. This study aimed to identify immunomodulatory effects and underlying mechanisms of AFNC using RAW264.7 macrophages and cyclophosphamide-induced immunosuppressed mice. As a result, AFNC-treated RAW264.7 macrophages elevated the production of IL-2, IL-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO). AFNC-enhanced inducible NO synthase expression via nuclear factor-κB signaling pathways. AFNC dose-dependently increased levels of IL-2, IL-6, TNF-α, IL-10, IL-12, interferon-γ, or IL-4 in the serum and spleen of immunosuppressed mice. Taken together, AFNC encourages the immune-enhancing activity through immunostimulatory cytokine production by activation of macrophage. Therefore, these results suggest that AFNC is useful for immunodeficiency-related disorders. PRACTICAL APPLICATIONS: In these days of prevalence of infectious diseases, individual immunity is very important. AFNC has the immune-enhancing effects through immunostimulatory cytokine production by activation of macrophage. Therefore, AFNC could be widely applied to ameliorate a variety of diseases caused by immunosuppression such as infectious diseases.

Dexamethasone Attenuates Oncostatin M Production via Suppressing of PI3K/Akt/NF-κB Signaling in Neutrophil-like Differentiated HL-60 Cells.

Oncostatin M (OSM) plays a role in various inflammatory reactions, and neutrophils are the main source of OSM in pulmonary diseases. However, there is no evidence showing the mechanism of OSM production in neutrophils. While dexamethasone (Dex) has been known to exert anti-inflammatory activity in various fields, the precise mechanisms of OSM downregulation by Dex in neutrophils remain to be determined. Here, we examined how OSM is produced in neutrophil-like differentiated HL-60 cells. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis were utilized to assess the potential of Dex. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation resulted in OSM elevation in neutrophil-like dHL-60 cells. OSM elevation induced by GM-CSF is regulated by phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor (NF)-kB signal cascades. GM-CSF stimulation upregulated phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Treatment with Dex decreased OSM levels as well as the phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Our findings show the potential of Dex in the treatment of inflammatory diseases via blocking of OSM.

Effects of Resveratrol on Thymic Stromal Lymphopoietin Expression in Mast Cells.

Background and objectives: Cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathogenesis of atopic diseases such as atopic dermatitis, allergic rhinitis, and asthma. Resveratrol (RSV) exerts various pharmacological effects such as antioxidant, anti-inflammatory, neuroprotective, and anticancer. Although, it has been verified the beneficial effects of RSV on various subjects, the effect of RSV on thymic stromal lymphopoietin (TSLP) regulation has not been elucidated. Materials and Methods: Here, we examined how RSV regulates TSLP in HMC-1 cells. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, Western blotting, and calcium assay were performed to evaluate the effect of RSV. Results: TSLP production and mRNA expression were reduced by RSV. RSV down-regulated nuclear factor-κB activation, IκBα phosphorylation as well as activation of receptor-interacting protein2 and caspase-1 in HMC-1 cells. In addition, RSV treatment decreased the up-regulation of intracellular calcium in HMC-1 cells. Conclusions: These results suggest that RSV might be useful for the treatment of atopic diseases through blocking of TSLP.

Chrysophanol, an anthraquinone from AST2017-01, possesses the anti-proliferative effect through increasing p53 protein levels in human mast cells.

OBJECTIVE: Natural products are well known as the source of drugs in the treatment of allergic inflammation. Chrysophanol, an anthraquinone from the AST2017-01 extract, showed a beneficial anti-inflammatory effect on activated human mast cells in our previous study. However, a regulatory effect of AST2017-01 and chrysophanol on mast cell proliferation induced by thymic stromal lymphopoietin (TSLP) remains unclear. The present study determined the anti-proliferative effect and the fundamental mechanism of AST2017-01 and chrysophanol in mast cells. METHODS: We evaluated an anti-proliferative effect of AST2017-01 and chrysophanol in TSLP-stimulated human mast cell line, HMC-1. RESULTS: Without cytotoxicity, AST2017-01 and chrysophanol decreased mast cells growth and Ki67 mRNA expression increased by TSLP. AST2017-01 and chrysophanol enhanced expressions of p53 and Bax, whereas inhibited expression of Bcl-2. AST2017-01 and chrysophanol restored caspase-3 activity which was decreased by TSLP. AST2017-01 and chrysophanol suppressed expressions of murine double minute-2 protein and phosphorylated-signal transducer and activator of transcription six which are associated with the regulation of p53 protein. AST2017-01 and chrysophanol decreased levels of interleukin (IL)-13, IL-6, and tumor necrosis factor-α. Moreover, AST2017-01 and chrysophanol reduced mRNA expressions of TSLP receptor and IL-7 receptor α. CONCLUSIONS: Therefore, this study proposes that AST2017-01 and chrysophanol may be promising candidates for the development of potent anti-inflammatory or health functional foods.