Chrysin Inhibits Lymphangiogenesis in Vitro.

The induction of lymphangiogenesis is an important process to promote cancer growth and cancer metastasis via the lymphatic system. Identifying the compounds that can prevent lymphangiogenesis for cancer therapy is urgently required. Chrysin, 5,7-dihydroxyflavone, a natural flavone extracted from Thai propolis, was used to investigate the effect on the lymphangiogenesis process of TR-LE, rat lymphatic endothelial cells. In this study, maximal nontoxic doses of chrysin on TR-LE cells were selected by performing a proliferation assay. The process of lymphangiogenesis in vitro was determined by cord formation assay, adhesion assay and migration assay. Chrysin at a nontoxic dose (25 µM) significantly inhibited cord formation, cell adhesion and migration of TR-LE cells when compared with the control group. We also found that chrysin significantly induced vascular endothelial growth factor C (VEGF-C) mRNA expression and nitric oxide (NO) production in TR-LE cells which was involved in decreasing the cord formation of TR-LE cells. In conclusion, we report for the first time that chrysin inhibited the process of lymphangiogenesis in an in vitro model. This finding may prove to be a natural compound for anti-lymphangiogenesis that could be developed for use in cancer therapy.

CXCL16 suppresses liver metastasis of colorectal cancer by promoting TNF-α-induced apoptosis by tumor-associated macrophages.

BACKGROUND: Inhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients. METHODS: We generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages. RESULTS: CXCL16 expression enhanced TNF-α-induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-κB-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF-α-induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF-α-induced apoptosis through the induction of M1 macrophages, which released TNF-α. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis. CONCLUSIONS: Collectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.

Immunostimulatory properties of heat-resistant RNA in a decoction of Glycyrrhizae Radix.

Research on the bioactive components of herbal medicines have been conducted mainly on the secondary metabolites of herbal plants. Accordingly, limited information is available on primary metabolites (carbohydrates, amino acids, lipids, and nucleic acids) and their biological effects. Here, we focused on the heat-resistant RNA of a decoction of Glycyrrhizae Radix and showed its immunostimulatory effects. The RNA activated NF-κB/AP-1 and induced TNF-α production in murine macrophages. Further analysis revealed that the RNA was around 90 nucleotides long. RNA sequencing (RNA-Seq) by next generation sequencing (NGS) showed that approximately 30% of the NGS reads were mapped to the genome of Glycyrrhiza uralensis, which is plant material of Glycyrrhizae Radix. Further analysis of the other 70% of reads indicated that the RNA contained RNA sequences that could be mapped to various microorganisms. Together, these results propose nucleic acids as a new research field in the bioactive components of herbal medicines.

Gomisin N enhances TNF-α-induced apoptosis via inhibition of the NF-κB and EGFR survival pathways.

Tumor necrosis factor (TNF-α) is a pleiotropic cytokine that plays an important role in the control of cell proliferation, differentiation, and apoptosis. TNF-α-induced apoptosis is limited by TAK1-mediated activation of NF-κB (mainly p65-p50 hetrodimer) signaling pathway. We have recently reported that TAK1 regulates phosphorylation of EGFR at Ser-1046/7 through p38 MAPK, which cooperates with NF-κB in TNF-α-induced apoptosis. The present study investigated the effect of gomisins A and N, dibenzocyclooctadiene lignans isolated from the fruit of Schisandra chinensis, on TNF-α-induced apoptosis in HeLa cells. Gomisins A and N strongly promoted TNF-α-induced cleavage of caspase-3 and PARP-1, which are key markers of apoptosis. We found that gomisin N, but not gomisin A, inhibited the TNF-α-induced activation of NF-κB by suppressing the activation of IKKα. Gomisin N also inhibited p38-mediated phosphorylation of the EGFR at Ser-1046/7 and subsequent endocytosis of EGFR, another prosurvival pathway. The findings suggested that gomisin N enhanced TNF-α-induced apoptosis by suppressing of NF-κB and EGFR signaling pathways.

Corrigendum to "Suppression of Dynamical Network Biomarker Signals at the Predisease State (Mibyou) before Metabolic Syndrome in Mice by a Traditional Japanese Medicine (Kampo Formula) Bofutsushosan".

[This corrects the article DOI: 10.1155/2020/9129134.].

A phagocytotic inducer from herbal constituent, pentagalloylglucose enhances lipoplex-mediated gene transfection in dendritic cells.

Antigen-presenting cells are key vehicles for delivering antigens in tumor immunotherapy, and the most potent of them are dendritic cells (DCs). Recent studies have demonstrated the usefulness of DCs genetically modified by lipofection in tumor immune therapy, although sufficient gene transduction into DCs is quite difficult. Here, we show that Paeoniae radix, herbal medicine, and the constituent, 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), have an attractive function to enhance phagocytosis in murine dendritic cell lines, DC2.4 cells. In particular, PGG in combination with lipofectin (LPF) enhanced phagocytic activity. Furthermore, PGG enhanced lipofection efficacy in DC2.4 cells, but not in colorectal carcinoma cell lines, Colon26. In other words, PGG synergistically enhanced the effect of lipofectin-dependent phagocytosis on phagocytic cells. Hence, according to our data, PGG could be an effective aid in lipofection using dendritic cells. Furthermore, these findings provide an expectation that constituents from herbal plant enhance lipofection efficacy.

Suppression of Dynamical Network Biomarker Signals at the Predisease State (Mibyou) before Metabolic Syndrome in Mice by a Traditional Japanese Medicine (Kampo Formula) Bofutsushosan.

Due to the increasing incidence of metabolic syndrome, the development of new therapeutic strategies is urgently required. One promising approach is to focus on the predisease state (so-called Mibyou in traditional Japanese medicine) before metabolic syndrome as a preemptive medical target. We recently succeeded in detecting a predisease state before metabolic syndrome using a mathematical theory called the dynamical network biomarker (DNB) theory. The detected predisease state was characterized by 147 DNB genes among a total of 24,217 genes in TSOD (Tsumura-Suzuki Obese Diabetes) mice, a well-accepted model of metabolic syndrome, at 5 weeks of age. The timing of the predisease state was much earlier than the onset of metabolic syndrome in TSOD mice reported to be at approximately 8-12 weeks of age. In the present study, we investigated whether the predisease state in TSOD mice can be inhibited by the oral administration of a Kampo formula, bofutsushosan (BTS), which is usually used to treat obese patients with metabolic syndrome in Japan, from 3 to 7 weeks of age. We found the comprehensive suppression of the early warning signals of the DNB genes by BTS at 5 weeks of age and later. Specifically, the standard deviations of 134 genes among the 147 DNB genes decreased at 5 weeks of age as compared to the nontreatment control group, and 80 of them showed more than 50% reduction. In addition, at 7 weeks of age, the body weight and blood glucose level were significantly lower in the BTS-treated group than in the nontreatment control group. The results of our study suggest a novel mechanism of BTS; it suppressed fluctuations of the DNB genes at the predisease state before metabolic syndrome and thus prevented the subsequent transition to metabolic syndrome. In conclusion, this study demonstrated the preventive and preemptive effects of a Kampo formula on Mibyou before metabolic syndrome for the first time based on scientific evaluation.

Shikonin induces an anti­tumor effect on murine mammary cancer via p38­dependent apoptosis.

Breast cancer is the most common malignancy in women. Apoptosis is important for tumor suppression and may delay cancer progression. It was found that shikonin induced apoptosis in 4T1 murine mammary cancer cells and MDA­MB­231 human breast cancer cells in vitro. Total p38 and c­Jun N­terminal kinase (JNK) levels were maintained in 4T1 cells, and p38 phosphorylation, but not JNK phosphorylation, was significantly increased. Caspase­3/7 activity was detected, which suggested that the p38 pathway, but not the JNK signaling pathway, induced apoptosis in 4T1 cells. The anti­tumor effects of shikonin on orthotopic mouse models were also examined. On day 7 after inoculation of 4T1 cells into mice, tumor volumes in the shikonin­treated and the control groups began to differ. On day 13, tumors were weighed, and shikonin was revealed to suppress tumor growth in the orthotopic 4T1 model in vivo. In conclusion, shikonin is a potential anti­tumor drug for breast cancer.

Identifying pre-disease signals before metabolic syndrome in mice by dynamical network biomarkers.

The establishment of new therapeutic strategies for metabolic syndrome is urgently needed because metabolic syndrome, which is characterized by several disorders, such as hypertension, increases the risk of lifestyle-related diseases. One approach is to focus on the pre-disease state, a state with high susceptibility before the disease onset, which is considered as the best period for preventive treatment. In order to detect the pre-disease state, we recently proposed mathematical theory called the dynamical network biomarker (DNB) theory based on the critical transition paradigm. Here, we investigated time-course gene expression profiles of a mouse model of metabolic syndrome using 64 whole-genome microarrays based on the DNB theory, and showed the detection of a pre-disease state before metabolic syndrome defined by characteristic behavior of 147 DNB genes. The results of our study demonstrating the existence of a notable pre-disease state before metabolic syndrome may help to design novel and effective therapeutic strategies for preventing metabolic syndrome, enabling just-in-time preemptive interventions.

Partial depletion of CD206-positive M2-like macrophages induces proliferation of beige progenitors and enhances browning after cold stimulation.

Beige adipocytes are an inducible form of thermogenic adipocytes that become interspersed within white adipose tissue (WAT) depots in response to cold exposure. Previous studies have shown that type 2 cytokines and M2 macrophages induce cold-induced browning in inguinal WAT (ingWAT) by producing catecholamines. Exactly how the conditional and partial depletion of CD206+ M2-like macrophages regulates the cold-induced browning of ingWAT, however, remains unknown. We examined the role of CD206+ M2-like macrophages in the cold-induced browning of WAT using genetically engineered CD206DTR mice, in which CD206+ M2-like macrophages were conditionally depleted. The partial depletion of CD206+ M2-like enhanced UCP1 expression in ingWAT, as shown by immunostaining, and also upregulated the expression of Ucp1 and other browning-related marker genes in ingWAT after cold exposure. A flow cytometry analysis showed that the partial depletion of CD206+ M2-like macrophages caused an increase in the number of beige progenitors in ingWAT in response to cold. Thus, we concluded that CD206+ M2-like macrophages inhibit the proliferation of beige progenitors and that the partial depletion of CD206+ M2-like macrophages releases this inhibition, thereby enhancing browning and insulin sensitivity.

Discovery of a sugar-based nanoparticle universally existing in boiling herbal water extracts and their immunostimulant effect.

Herbal medicine is mainly prepared from boiling herbal water extracts. Many epoch-making immunosuppressant drugs, such as glycyrrhizic acid (old example) and FTY720 (current example), were developed from herbal secondary metabolites in the boiling water extract by partition with organic solvents. However, few immunostimulants have been discovered by this method. Instead of the usual method, we aimed to find a novel immunostimulant component by two unique methods in the research of herbal medicine: ultracentrifugation and electron microscopy. The immunostimulant was not a secondary metabolite, as expected, but the structure was a nanoparticle formed by a polysaccharide. In addition, we clarified the immune effect of the nanoparticle. Intake of the nanoparticle by phagocytosis resulted in immunostimulant effects by increasing the genes and proteins of inflammatory cytokines in macrophage cells. The immunostimulant effects were inhibited by a phagocytosis inhibitor, cytochalasin D. To the best of our knowledge, this study is the first to describe the discovery of a nanoparticle in boiling herbal water extracts and its immunostimulant properties. This study will provide additional understanding of the efficacy of herbal medicine, in that the immunostimulant nanoparticle universally exists in boiling herbal water extracts. Thus, traditional herbal medicine may be an oldest known nanomedicine. Furthermore, this study suggests that the immunostimulant nanoparticle simply can be obtained from herbal medicine only by ultracentrifugation. We hope that this simple strategy will substantially contribute to drug development, including vaccine adjuvant, in the future.

Immune adjuvant effect of Juzentaihoto, a Japanese traditional herbal medicine, on tumor vaccine therapy in a mouse model.

Japanese traditional herbal medicine (Kampo) have been used to improve the general physical condition after surgery and to mitigate the side effects of radiation and chemotherapy in tumor patients. Juzentaihoto (JTT) consists of ten medical herbs, and is also called Shi-Quan-Da-Bu-Tang in Chinese herbal medicine. Among Kampo medicines, JTT has especially gained attention as a biological response modifier. Currently, clinical trials of various tumor vaccine therapies are being performed world-wide. However, tumor antigens that are inoculated as vaccines do not have high immunogenicity; thus, it is difficult to obtain an effective therapeutic effect. Thus, it is necessary to develop a tumor vaccine adjuvant that is more potent and very safe. In the present study, we examined the efficacy of JTT as an oral adjuvant when given together with tumor vaccines. As a result, JTT enhanced the phagocytic ability of OVA antigen and the presentation ability of OVA antigen in dendritic cells in vitro. Furthermore, tumor growth was markedly decreased, and the survival period was significantly prolonged in mice inoculated with mouse lymphoma, which is expressed with tumor model antigen. In conclusion, these findings suggest that JTT can be used with tumor vaccines as an immune adjuvant.

Inhibitory effect of Moutan Cortex aqueous fraction on mast cell-mediated allergic inflammation.

Moutan Cortex and its major compounds have been shown to possess various biological activities, including anti-inflammatory properties. However, the effects of Moutan Cortex aqueous fraction (MCA) and its molecular mechanisms have yet to be elucidated. In this study, we attempted to evaluate the effects of MCA on mast cell-mediated allergy inflammation in vitro and in vivo compared with major Moutan Cortex compounds. Thus, we examined the anti-inflammatory effects of a water extract of Moutan Cortex by comparing the inhibition of ß-hexosaminadase and tumor necrosis factor-α (TNF-α) release in an aqueous fraction with other major compounds of Moutan Cortex. The inhibitory mechanism of MCA was investigated by western blotting in IgE-mediated DNP-BSA-stimulated RBL-2H3 cells. We confirmed the pharmacological effects of MCA on compound 48/80-induced allergic reactions in a mouse model by assessing scratching behavior and passive cutaneous anaphylaxis (PCA)-like reaction. Consequently, MCA inhibited IgE-mediated DNP-BSA-induced ß-hexosaminadase and TNF-α release via inactivation of p38, ERK, Akt, and NF-κB in RBL-2H3 cells. MCA reduced compound 48/80-induced PCA reaction and scratching behavior in mice. This inhibitory effect of MCA is more potent than major compounds of Moutan Cortex. In conclusion, our results suggest that MCA has more potential in the treatment of allergic inflammatory diseases compared to other major compounds of Moutan Cortex.

Enhancement of Lymphangiogenesis In Vitro via the Regulations of HIF-1α Expression and Nuclear Translocation by Deoxyshikonin.

The objectives of this study were to determine the effects of deoxyshikonin on lymphangiogenesis. Deoxyshikonin enhanced the ability of human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) to undergo time-dependent in vitro cord formation. Interestingly, an opposite result was observed in cells treated with shikonin. The increased cord formation ability following deoxyshikonin treatment correlated with increased VEGF-C mRNA expression to higher levels than seen for VEGF-A and VEGF-D mRNA expression. We also found that deoxyshikonin regulated cord formation of HMVEC-dLy by increasing the HIF-1 α mRNA level, HIF-1 α protein level, and the accumulation of HIF-1 α in the nucleus. Knockdown of the HIF-1 α gene by transfection with siHIF-1 α decreased VEGF-C mRNA expression and cord formation ability in HMVEC-dLy. Deoxyshikonin treatment could not recover VEGF-C mRNA expression and cord formation ability in HIF-1 α knockdown cells. This indicated that deoxyshikonin induction of VEGF-C mRNA expression and cord formation in HMVEC-dLy on Matrigel occurred mainly via HIF-1 α regulation. We also found that deoxyshikonin promoted wound healing in vitro by the induction of HMVEC-dLy migration into the wound gap. This study describes a new effect of deoxyshikonin, namely, the promotion of cord formation by human endothelial cells via the regulation of HIF-1 α . The findings suggest that deoxyshikonin may be a new drug candidate for wound healing and treatment of lymphatic diseases.

The long-term effects of a kampo medicine, juzentaihoto, on maintenance of antibody titer in elderly people after influenza vaccination.

We have performed a broad-ranging analysis of the adjuvant effect of a Kampo medicine, juzentaihoto (JTT), on influenza vaccination in a multicenter randomized controlled trial. In this study, the enhancing effect of JTT on antibody titer after influenza vaccination was studied for 28 weeks in elderly people who were in the high-risk group for influenza infection. In total, 91 subjects over 65 years old were recruited from four long-term-care facilities located in Chiba, Gunma, and Toyama prefectures in Japan. Participants were randomly assigned to the JTT and the control groups. Blood samples were taken at 4 weeks before vaccination, at the time of vaccination, and then at 4, 8, 12, and 24 weeks after vaccination. The hemagglutination inhibition (HI) titers against A/California/7/2009 (H1N1), A/Victoria/210/2009 (H3N2), and B/Brisbane/60/2008 were then manually measured. A significant increase in HI titer against H3N2 was observed at week 8 after vaccination in the JTT group compared with the control group (P = 0.0229), and the HI titer of the JTT group significantly increased from 4 to 24 weeks (P = 0.0468), compared with the control group. In conclusion, our results indicated that JTT increased and prolonged antibody production against A/Victoria/210/2009 (H3N2), in particular, after influenza vaccination.

Bufotalin sensitizes death receptor-induced apoptosis via Bid- and STAT1-dependent pathways.

Tumor necrosis factor-alpha (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) are apoptosis-inducing ligands that stimulate death receptors. In this study, we investigated the effects of bufotalin, a major compound in toad venom, on sensitizing TNF-α and TRAIL-induced apoptosis of HeLa cells. Bufotalin promoted death receptor-mediated cell death, especially TRAIL-induced apoptosis, through activation of caspase-3 and PARP-1. Mitochondrial Bid-dependent pathway was activated in TNF-α-induced cell death. Cotreatment of bufotalin with TRAIL resulted in the downregulation of anti-apoptotic proteins, including Bcl-XL, Mcl-1, survivin and XIAP, and the up-regulation of MAPKs and TRAIL receptor DR5. In addition, phosphorylation of STAT1 was strongly inhibited by bufotalin. Moreover, DR5 expression was induced by knocking down the STAT1 expression. Moreover, the TRAIL-induced apoptotic response was promoted by STAT1 siRNA. Our results demonstrated that bufotalin is a powerful sensitizer of death receptor-induced apoptosis in cancer cells.