Dihydroxystilbenes prevent azoxymethane/dextran sulfate sodium-induced colon cancer by inhibiting colon cytokines, a chemokine, and programmed cell death-1 in C57BL/6J mice.

The incidence of colon cancer increased worldwide in 2019 and its treatment is urgent from a quality of life perspective. A relationship has been reported between elevated numbers of tumor-associated macrophages (TAMs) in the tumor microenvironment and a poor prognosis in cancer patients, and M2 TAMs have been shown to promote tumor growth by immunosuppression through the stimulation of programmed death-1 (PD-1, an immune check point receptor), interleukin (IL)-1ß, and monocyte chemoattractant protein (MCP)-1. We herein examined the effects of three synthetic dihydroxystilbenes (2,3-, 3,4-, and 4,4'-dihydroxystilbenes) on colon carcinogenesis, colon tumor growth, and colon cytokines (IL-1ß, IL-6, and tumor necrosis factor (TNF)-α), a chemokine (MCP-1), vascular endothelial growth factor (VEGF), and PD-1 levels in azoxymethane (AOM) plus dextran sulfate sodium (DSS)-treated C57BL/6J mice. The three dihydroxystilbenes inhibited colon carcinogenesis and tumor growth as well as increases in colon IL-1ß, IL-6, MCP-1, and PD-1 levels in AOM/DDS-treated mice (in vivo). The three dihydroxystilbenes also suppressed COX-2 expression in colon tumors (in vivo). The results obtained also revealed that the three dihydroxystilbenes inhibited PD-1 elevations in M2-THP-1 macrophages (in vitro). Therefore, the inhibition of AOM/DSS-induced colon carcinogenesis and colon tumor growth by 2,3-, 3,4-, and 4,4'-dihydroxystilbenes appears to be due to the suppression of M2 TAM differentiation and activation and PD-1 expression (immunosuppression) via reductions in COX-2 expression levels in the colon tumor microenvironment.

Suksdorfin Promotes Adipocyte Differentiation and Improves Abnormalities in Glucose Metabolism via PPARγ Activation.

Although the Apiaceae herb family has been traditionally used for the management of type 2 diabetes, its molecular mechanism has not been clarified. Coumarin derivatives, which are abundant in plants of the Apiaceae family, were evaluated for their effects on adipogenesis. We found that suksdorfin significantly promoted adipocyte differentiation and enhanced production of adiponectin, an anti-diabetic adipokine. We also demonstrated that suksdorfin activates peroxisome proliferator-activated receptor gamma (PPARγ), a master regulator of adipogenesis. Furthermore, we showed metabolic disorders in obese diabetic KK-Ay mice were attenuated by suksdorfin feeding. Suksdorfin intake induced adipocyte miniaturization and increased expression levels of PPARγ target genes related to adipocyte differentiation. These results indicated that suksdorfin induces adipogenesis in white adipose tissue (WAT) via the activation of PPARγ, leading to improvement of obesity-induced metabolic disorders. Therefore, suksdorfin-mediated amelioration of WAT dysfunctions might be responsible for the anti-diabetic effects of traditional herbal medicine therapy with Apiaceae.

Xanthoangelol and 4-hydroxyderrcin suppress obesity-induced inflammatory responses.

OBJECTIVE: Obesity-induced inflammation plays a pivotal role in the pathogenesis of insulin resistance and type 2 diabetes. Xanthoangelol (XA) and 4-hydroxyderrcin (4-HD), phytochemicals extracted from Angelica keiskei, have been reported to possess various biological properties. Whether XA and 4-HD alleviate obesity-induced inflammation and inflammation-induced adipocyte dysfunction was investigated. METHODS: For the in vitro study, a co-culture system composed of macrophages and adipocytes and macrophages stimulated with conditioned medium derived from fully differentiated adipocytes was conducted. For the in vivo study, mice were fed a high-fat diet supplemented with XA for 14 weeks. RESULTS: XA and 4-HD suppressed inflammatory factors in co-culture system. Moreover, treatment of RAW macrophages with XA and 4-HD moderated the suppression of uncoupling protein 1 promoter activity and gene expression in C3H10T1/2 adipocytes, which was induced by conditioned medium derived from LPS-stimulated RAW macrophages. Also, XA and 4-HD inhibited c-Jun N-terminal kinase phosphorylation, nuclear factor-κB, and activator protein 1, the last two being transcription activators in activated macrophages. Furthermore, in mice fed the high-fat diet, XA reduced inflammatory factors within the white adipose tissue. CONCLUSIONS: These results suggest that XA and 4-HD might be promising phytochemicals to suppress obesity-induced inflammation and inflammation-induced adipocyte dysfunction.

4-Hydroxyderricin, as a PPARγ Agonist, Promotes Adipogenesis, Adiponectin Secretion, and Glucose Uptake in 3T3-L1 Cells.

Adipocyte differentiation plays a pivotal role in maintaining the production of small-size adipocytes with insulin sensitivity, and impaired adipogenesis is implicated in insulin resistance. 4-Hydroxyderricin (4-HD), a phytochemical component of Angelica keiskei, possesses diverse biological properties such as anti-inflammatory, antidiabetic, and antitumor. In the present study, we investigated the effects of 4-HD on adipocyte differentiation. 4-HD promoted lipid accumulation in 3T3-L1 cells, upregulated both peroxisome proliferator-activated receptor (PPAR)-γ mRNA and protein expression, and acted as a ligand for PPARγ in the luciferase assay. Moreover, 4-HD increased the mRNA and protein expression levels of adiponectin. Additionally, it promoted insulin-dependent glucose uptake into 3T3-L1 adipocytes and increased Akt phosphorylation and glucose transporter (GLUT) 4 mRNA expression. In summary, these findings suggest that 4-HD, which promoted adipogenesis and insulin sensitivity in 3T3-L1 cells, might be a phytochemical with potent insulin-sensitizing effects.

Antitumor and Antimetastatic Activity of Synthetic Hydroxystilbenes Through Inhibition of Lymphangiogenesis and M2 Macrophage Differentiation of Tumor-associated Macrophages.

An increase in tumor-associated macrophages (TAMs) around the tumor microenvironment has been closely associated with a poor prognosis in patients with cancer, and M2 TAMs promote tumor growth and tumor metastasis by stimulating angiogenesis or lymphangiogenesis in tumors. We herein examined the effects of nine synthetic hydroxystilbenes on M2 macrophage activation and differentiation, and three selected dihydroxystilbenes on vascular endothelial cell growth factor (VEGF)-C-induced tube formation in human lymphatic endothelial cells (HLECs) (in vitro). We also investigated the antitumor and antimetastatic effects of three synthetic dihydroxystilbenes in LM8-bearing mice in vivo. The three selected synthetic stilbenes (at concentrations of 5, 10, 25, and 50 µM) inhibited the production of interleukin-10 and monocyte chemoattractant protein-1 in M2 macrophages, but promoted that of transforming growth factor-ß1. The three dihydroxystilbenes (at concentrations of 10-50 µM) inhibited the phosphorylation of signal transducer and activator of transcript 3 without affecting its expression in the differentiation of M2 macrophages. Furthermore, the 2,3- and 4,4'-dihydroxystilbene inhibited VEGF-C-induced lymphangiogenesis in HLECs. Both 2,3- and 4,4'-dihydroxystilbene (at 10 and 25 mg/kg, twice daily) inhibited tumor growth and metastasis to the lung in mice. These results suggested that the antitumor and antimetastatic effects of 2,3- and 4,4'-dihydroxystilbene were partly due to anti-lymphangiogenesis, and the regulation of M2 macrophage activation and differentiation.

Antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderricin isolated from Angelica keiskei roots through the inhibited activation and differentiation of M2 macrophages.

BACKGROUND: Tumor growth and metastasis have been closely associated with the M2 macrophage-induced activation of tumor-associated macrophages (TAMs). PURPOSE: The antitumor and antimetastatic actions of xanthangelol and 4-hydroxyderricin on the role of M2 macrophages in the TAMs of highly metastatic osteosarcoma LM8-bearing mice have not yet been fully elucidated. In order to clarify the mechanisms underlying the antitumor and antimetastatic actions of the above chalcones, we performed in vivo and in vitro studies. STUDY DESIGN: The antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderricin were examined in vivo and the effects on M2 macrophage differentiation and activation were examined in vitro. METHODS: We examined the antitumor and antimetastatic effects of xanthoangelol and 4-hydroxyderricin on highly metastatic osteosarcoma LM8-bearing mice (in vivo). Further, we examined their effects on the differentiation of interleukin (IL)-4 plus IL-13-induced M2 macrophages and activation of IL-4 plus IL13-induced M2 macrophages (in vitro). We also investigated the expression and phosphorylation of signal transducer and activator of transcript 3 (Stat 3) in the differentiation process of M2-polarized macrophages (in vitro). RESULTS: Xanthoangelol or 4-hydroxyderricin (25 or 50 mg/kg, twice daily) inhibited tumor growth, metastasis to the lung and liver, and TAM expression in tumors. In addition, xanthoangelol (10, 25 or 50 µM) and 4-hydroxyderricin (5, 10, 25 or 50 µM) inhibited the production of IL-10 and monocyte chemoattractant protein (MCP)-1 in M2-polarized macrophages. This result indicated that xanthoangelol and 4-hydroxyderricin inhibited the activation of M2 macrophages. Furthermore, xanthoangelol (5-50 µM) inhibited the phosphorylation of Stat 3 without affecting the expression of the Stat 3 protein in the differentiation process of M2 macrophages, which indicated that these chalcones inhibited the differentiation of M2 macrophages. CONCLUSION: These findings suggested that the antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderrcin might be attributed to the regulated activated TAMs through the inhibition of activation and differentiation of M2 macrophages in the tumor microenvironment.

Synergistic antitumor effect of a combination of paclitaxel and carboplatin with nobiletin from Citrus depressa on non-small-cell lung cancer cell lines.

Non-small-cell lung carcinomas do not sufficiently respond to cancer chemotherapeutic drugs. Combination effects of cancer chemotherapy drugs (paclitaxel and carboplatin) with nobiletin or powdered Shiikuwasha extract from Citrus depressa were examined by isobologram and combination index analyses. It was demonstrated that the combination generated a synergistic inhibitory effect against the proliferation of the human non-small-cell lung carcinoma cell lines A549 and H460 and that of the two chemotherapy drugs, paclitaxel was responsible for this synergistic effect. Furthermore, the percentage of apoptotic cells was decreased with increasing rates of nobiletin to paclitaxel and carboplatin. These findings were considered to be attributed to the ability of nobiletin to regulate cells in the G1 phase, which escaped cell death initiated by paclitaxel and carboplatin. An antitumor activity assay showed that this combination significantly suppressed the growth of subcutaneous A549 tumor xenografts in nude mice.

Bio-imaging of hydroxyl radicals in plant cells using the fluorescent molecular probe rhodamine B hydrazide, without any pretreatment.

Rhodamine B hydrazide can be used to detect hydroxyl radicals in plant cells. RBH was easily inserted into plant cells without any pretreatment, and specifically reacted with intracellular hydroxyl radicals produced by antimycin A. RBH will be a powerful tool for detecting hydroxyl radicals in plant cells.

Anti-tumor effects of various furocoumarins isolated from the roots, seeds and fruits of Angelica and Cnidium species under ultraviolet A irradiation.

We examined the effects on cell proliferation of 10 methoxyfurocoumarins and 7 dihydrofurocumarins isolated from Umbelliferae medicinal plants, and their mechanisms of action against B16F10 melanoma cells or in melanin-possessing hairless mice implanted with B16F10 melanoma cells, under UVA irradiation. Furocoumarins having a methoxy group, such as bergapten (1), xanthotoxin (2), phellopterin (4), byakangelicin (6), neobyakangelicin (8), isobergapten (9) and sphondin (10), showed anti-proliferative activity and caused G2/M arrest at concentrations of 0.05-15.0 µM. The 7 dihydrofurocoumarins had no effect. UVA plus 1, 2, 4, 6 and sec-O-acetylbyakagelicin (7), having one methoxy group at the C-5 position and a linear-type conformation, reduced tumor growth and final tumor weight in B16F10-bearing mice at 0.5 or 1.0 mg/kg (intraperitoneal injection). UVA plus 1 and 2 increased Chk1 phosphorylation and decreased cdc2 (Thr 161) phosphorylation in the melanoma cells. The anti-tumor actions of UVA plus furocoumarins having a methoxy group might be due to the arrest of the cell cycle at G2/M through an increase in phospho-Chk1 and reduction in phospho-cdc2.

Sinodielide A exerts thermosensitizing effects and induces apoptosis and G2/M cell cycle arrest in DU145 human prostate cancer cells via the Ras/Raf/MAPK and PI3K/Akt signaling pathways.

Sinodielide A (SA) is a naturally occurring guaianolide, which is isolated from the root of Sinodielsia yunnanensis. This root, commonly found in Yunnan province, is used in traditional Chinese medicine as an antipyretic, analgesic and diaphoretic agent. A number of studies have reported that agents isolated from a species of Umbelliferae (Apiaceae) have antitumor activities. We previously reported, using combined treatments with this medicinal herb and hyperthermia at various temperatures, an enhanced cytotoxicity in the human prostate cancer androgen­independent cell lines, PC3 and DU145, and analyzed the related mechanisms. In the present study, we investigated the effects of treatment with SA prior to hyperthermia on the thermosensitivity of DU145 cells, and the mechanisms related to the induction of apoptosis and G(2)/M cell cycle arrest via the activation of extracellular-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathways, as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. Cells were exposed to hyperthermia alone (40-44˚C) or hyperthermia in combination with SA. Lethal damage to cells treated with mild hyperthermia (40 or 42˚C) for up to 6 h was slight; however, hyperthermia in combination with SA synergistically enhanced thermosensivity. Lethal damage to cells treated with acute hyperthermia (43 or 44˚C) was more severe, but these effects were also enhanced and were more significant by the combined treatment with SA. The kinetics of apoptosis induction and cell cycle distribution were analyzed by flow cytometry. In addition, the levels of ERK1/2, JNK and Akt were determined by western blot analysis. The incidence of apoptotic cells after treatment with SA (20.0 µM) at 37˚C for 4 h, hyperthermia (44˚C) alone for 30 min, and the combination in sequence were examined. The sub-G1 division (%) in the diagram obtained by flow cytometry was applied to that assay. The percentage of apoptotic cells (10.53±5.02%) was higher at 48 h as compared to 0, 12 and 24 h after treatment. The distribution of DU145 cells in the G2/M cell cycle phase was markedly increased after 24 h of heating at 44˚C and after the combined treatment with heating and SA. The phosphorylation of ERK1/2 was reduced following treatment with heating and SA, while the levels of phosphorylated JNK (p-JNK) were markedly increased immediately after heating at 44˚C and when heating was combined with SA. By contrast, the levels of phosphorylated Akt (p-Akt) were immediately increased only after heating at 44˚C. Thus, we concluded that SA exerts its thermosensitizing effects on DU145 cells by inhibiting the activation of the MAPK/ERK1/2 and PI3K/Akt signaling pathways.

In vitro and in vivo antiproliferative effect of a combination of ultraviolet-A and alkoxy furocoumarins isolated from Umbelliferae medicinal plants, in melanoma cells.

We examined the effects of six furocoumarins with alkoxy groups at the C-5 or C-8 position isolated from Umbelliferae medicinal plants on cell proliferation, and their mechanisms of action against B16F10 melanoma cells or in melanin-possessing hairless mice implanted with B16F10 cells, under UVA irradiation. Three furocoumarins with an alkoxy group at C-5, isoimperatorin (1), oxypeucedanin (2) and oxypeucedanin hydrate (3), showed antiproliferative activity and caused G2/M arrest at concentrations of 0.1-10.0 µm. Furthermore, three furocoumarins with an alkoxy group at C-8, imperatorin (4), heraclenin (5) and heraclenol (6), inhibited the proliferation of melanoma cells and cell cycle at G2/M at concentrations of 0.1-1.0 µm. UVA plus 1, 2, 3, 4 and 6 reduced tumor growth and final tumor weight in B16F10-bearing mice at a dose of 0.3, 0.5 or 1.0 mg kg(-1) (intraperitoneal injection). UVA plus 1, 3 and 6 increased Chk1 phosphorylation and reduced cdc2 (Thr 161) phosphorylation in melanoma cells. We suggest that the antitumor actions of UVA plus furocoumarins with an alkoxy group at C-5 or C-8 were due to G2/M arrest of the cell cycle by an increase in phosphor-Chk1 and decrease in phospho-cdc2.

Effects of furanocoumarins in Kampo extract-based medicines on rat intestinal absorption of CYP3A and P-glycoprotein substrate drugs in vivo.

While a great deal of information of drug-drug interactions is known, most concern Western drugs. Relatively little is known of the interactions between Western drugs and traditional drugs such as Kampo extract medicines (Japanese medicines modified from traditional Chinese medicines). This study investigated the effects of the marketed Kampo extract medicines, Senkyu-cha-cho-san and Sokei-kakketsu-to, on the intestinal absorption of CYP or P-glycoprotein (P-gp) in vivo. Midazolam, a CYP3A substrate drug, or talinolol, a P-gp substrate drug, was orally administered to rats with each of these Kampo extract medicines. Senkyu-cha-chosan or Sokei-kakketsu-to administered as a standard regimen did not obviously affect Cmax and area under the curve (AUC) of midazolam, although both Kampo extract medicines contained notopterol, a potent CYP3A4 inhibitor in vitro. The results implied a lack of potent drug-drug interactions between both Kampo extract medicines and CYP3A substrate drugs. Concomitant administration of each Kampo extract medicine unexpectedly showed the tendency to decrease Cmax and AUC of talinolol. Decreased intestinal absorption of talinolol might be caused, not by the inhibition of P-gp, but by the inhibition of organic anion transporting peptides by both Kampo extract medicines.

Anti-tumor activity of new orally bioavailable 2-amino-5-(thiophen-2-yl)benzamide-series histone deacetylase inhibitors, possessing an aqueous soluble functional group as a surface recognition domain.

New orally bioavailable 5-(thiophen-2-yl)-substituted 2-aminobenzamide-series histone deacetylase inhibitors were synthesized. These compounds possess a morpholine or piperadine-derived moiety as an aqueous soluble functional group. Among them, 8b, having a 4-ethyl-2,3-dioxopiperazine-1-carboxamide group as a surface recognition domain, showed promising inhibitory activities against HCT116 cell growth and HDAC1/2. Notably, unlike MS-275, this compound did not induce apoptosis in the cell cycle tests. We therefore conducted antitumor tests of 8b and MS-275 against HCT116 cell xenografts in nude mice. Compound 8b reduced the volume of tumor mass to T/C: 60% and 47% at 45 and 80mg/kg over 16days, respectively. These values were comparable to the rate (T/C: 51% at 45mg/kg) for MS-275. Furthermore, 8b, at neither 45 nor 80mg/kg, induced the weight loss which was observed in the mice given MS-275 at 45mg/kg.

[Chemical constituents from roots of Peucedanum praeruptorum (V)].

OBJECTIVE: To study the chemical constituents in roots of Peucedanum praeruptorum from Henan. METHOD: The constituents were isolated by column chromatography on silica gel and ODS, and identified by NMR, MS spectroscopic methods. RESULT: Six compounds, Pd-I b(1), marmesinsin (2), rutarin (3), isorutarin (4), 4H-1-benzopyran-4-one, 5-hydroxy-6-methoxy-2-phenyl-7-O-alpha-D-glucuronyl methyl ester (5), 4H-1-benzopyran-4-one, 5-hydroxy-6-methoxy-2-phenyl-7-O-alpha-D-glucuronyl acid (6) were isolated and identified. CONCLUSION: Compound 5 was a new compound, compounds 5 and 6 were isolated from Peucedanum plant for the first time.

Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production.

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNFα-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions.

Inhibitory effects of furanocoumarin derivatives in Kampo extract medicines on P-glycoprotein at the blood-brain barrier.

Furanocoumarin derivatives, known as components of grapefruit juice, showing inhibitory effects against P-glycoprotein (P-gp) in the intestine are also contained in the plants of rutaceae and umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of byakangelicol and rivulobirin A, known as furanocoumarins showing P-gp inhibitory effect using Caco-2 monolayer, against P-gp at the blood-brain barrier (BBB) under both in vitro and in vivo conditions. First we studied the membrane permeability of furanocoumarins to clarify whether they can be absorbed from the intestine. Both furanocoumarins showed high permeability through the Caco-2 monolayer, suggesting that they can easily reach the systemic circulation after oral administration. Then, we evaluated the effect of these furanocoumarins on the uptake of calcein acetoxymethyl ester (calcein-AM), a P-gp substrate, into bovine brain microvascular endothelial cells (BBMEC). Both furanocoumarins significantly increased the uptake amount of calcein-AM into BBMEC by the inhibition of P-gp at the BBB in vitro. Next we also investigated the P-gp inhibitory effect of these furanocoumarins at the rat BBB in vivo using verapamil as a P-gp substrate. Both furanocoumarins increased the B/P ratio of verapamil compared to the control, even under in vivo conditions; however, the extent of the inhibitory effect was much lower than in vitro condition. In conclusion, byakangelicol and rivulobirin A may inhibit P-gp expressed at the BBB even under in vivo conditions. Further studies using Kampo extract medicines under in vivo condition are necessary for safe drug therapy.

New microtubule polymerization inhibitors comprising a nitrooxymethylphenyl group.

We have designed cancer antiproliferative compounds, starting from aniline or phenol derivative, which comprise one or two nitrooxymethylphenyl groups as do the hybrid drugs NCX4040 and NCX530. Compound 2a with p-nitrooxymethylbenzoyl-oxy and -amino groups as well as 8a with a p-nitrooxymethylbenzoylamino group showed more promising effects than NCX4040 against human colon and breast cancer cells. Since 2a and 8a, but not NCX4040, arrested human colon carcinoma HCT116 cells in the M phase, the former two compounds may inhibit cell growth differently from NCX4040. Merged images of immunofluorescence-stained α-tubulin and Hoechst-stained nuclei in human fibrosarcoma HT1080 cells showed that 2a and 8a disrupted microtubule formation just as did vincristine, the tubulin polymerization inhibitor. In experiments in vivo, the intraperitoneal administration of 8a at 80 mg/kg/day reduced the growth of HCT116 xenografts in nude mice to T/C 55%.

Four novel furanocoumarin glucosides, candinosides A, B, C and D, from Heracleum candicans Wall.

Four novel furanocoumarin glucosides, candinosides A, B, C and D (1-4), were isolated from the roots of Heracleum candicans Wall. Their structures were established using chemical and spectral methods.

Five condensed furanocoumarins from the root of Heracleum candicans Wall.

Three new spirotrifuranocoumarins, canditririns C-E (1-3), a new spirotetrafuranocoumarin, canditetrarin A (4), and a new tetrafuranocoumarin, canditetrarin B (5), were isolated from the roots of Heracleum candicans Wall. Their structures were established using spectral methods.