Glycyrrhizin Attenuates Carcinogenesis by Inhibiting the Inflammatory Response in a Murine Model of Colorectal Cancer.

Glycyrrhizin (GL), an important active ingredient of licorice root, which weakens the proinflammatory effects of high-mobility group box 1 (HMGB1) by blocking HMGB1 signaling. In this study, we investigated whether GL could suppress inflammation and carcinogenesis in an azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced murine model of colorectal cancer. ICR mice were divided into four groups (n = 5, each)-control group, GL group, colon cancer (CC) group, and GL-treated CC (CC + GL) group, and sacrificed after 20 weeks. Plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured using an enzyme-linked immunosorbent assay. The colonic tissue samples were immunohistochemically stained with DNA damage markers (8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxy-guanosine), inflammatory markers (COX-2 and HMGB1), and stem cell markers (YAP1 and SOX9). The average number of colonic tumors and the levels of IL-6 and TNF-α in the CC + GL group were significantly lower than those in the CC group. The levels of all inflammatory and cancer markers were significantly reduced in the CC + GL group. These results suggest that GL inhibits the inflammatory response by binding HMGB1, thereby inhibiting DNA damage and cancer stem cell proliferation and dedifferentiation. In conclusion, GL significantly attenuates the pathogenesis of AOM/DSS-induced colorectal cancer by inhibiting HMGB1-TLR4-NF-κB signaling.

Glycyrrhizin ameliorates melanoma cell extravasation into mouse lungs by regulating signal transduction through HMGB1 and its receptors.

Metastasis, which accounts for the majority of all cancer-related deaths, occurs through several steps, namely, local invasion, intravasation, transport, extravasation, and colonization. Glycyrrhizin has been reported to inhibit pulmonary metastasis in mice inoculated with B16 melanoma. This study aimed to identify the mechanism through which glycyrrhizin ameliorates the extravasation of melanoma cells into mouse lungs. Following B16 melanoma cell injection, mice were orally administered glycyrrhizin once every two days over 2 weeks; lung samples were then obtained and analyzed. Blood samples were collected on the final day, and cytokine plasma levels were determined. We found that glycyrrhizin ameliorated the extravasation of melanoma cells into the lungs and suppressed the plasma levels of interleukin-6, tumor necrosis factor-α, and transforming growth factor-ß. Furthermore, glycyrrhizin ameliorated the lung tissue expression of high mobility group box-1 protein (HMGB1), receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)-4, RAS, extracellular signal-related kinase, NF-κB, myeloid differentiation primary response 88, IκB kinase complex, epithelial-mesenchymal transition markers, and vascular endothelial growth factor-A. Our study demonstrates that glycyrrhizin ameliorates melanoma metastasis by regulating the HMGB1/RAGE and HMGB1/TLR-4 signal transduction pathways.

Licochalcone A Inhibits BDNF and TrkB Gene Expression and Hypoxic Growth of Human Tumor Cell Lines.

Hypoxic cellular proliferation is a common feature of tumor cells and is associated with tumor progression. Therefore, the inhibition of hypoxic cellular proliferation is expected to regulate malignancy processes. Licochalcone A (LicA) is known to show inhibitory effects on cell growth in normoxia, but it is unclear whether LicA exerts similar effects in hypoxia. Here, we studied the inhibitory activity of LicA in the hypoxic cellular proliferation of tumor cells and its molecular mechanism using human cell lines derived from various tumors including neuroblastoma and colorectal cancer. LicA inhibited cell growth at a 50% inhibitory concentration between 7.0 and 31.1 µM in hypoxia. LicA significantly suppressed hypoxic induction of tropomyosin receptor kinase B (TrkB) gene expression at the transcription level. LicA also downregulated mRNA levels of the TrkB high-affinity ligand brain-derived neurotrophic factor, but not neurotrophin-4, another TrkB ligand, or glyceraldehyde-3-phosphate dehydrogenase, indicating that the inhibitory activity of LicA is selective. Since both LicA-treatment and TrkB-knockdown decreased activation of protein kinase B in hypoxia, LicA exerts its inhibitory effect against hypoxic cell growth through inhibition of the TrkB-AKT axis. These results suggest that LicA has therapeutic potential for malignant tumors including neuroblastoma and colorectal cancer.

Field Survey of Glycyrrhiza Plants in Central Asia (4). Characterization of G. glabra and G. bucharica Collected in Tajikistan.

The characteristics of 2 Glycyrrhiza plants, G. glabra and G. bucharica (=Meristotropis bucharica), were investigated in Tajikistan. The glycyrrhizin content in the underground parts of G. glabra varied from 2.56 to 9.29% of the dry weight, and the content of glabridin, a species-specific flavonoid of G. glabra, varied from 0.09 to 0.92% of the dry weight. Seeds of G. glabra plants from Tajikistan were cultivated for 3 years in Japan, and the glycyrrhizin content of the harvested roots ranged from 0.75 to 1.82% of the dry weight. In addition, HPLC analysis of leaf extracts indicated that the G. glabra plants collected in Tajikistan could be divided into various types, according to the flavonoid contents of the leaves. The endemic G. bucharica was also collected. A phylogenetic tree of rbcL nucleotide sequences from various Glycyrrhiza plants indicated that G. bucharica was closely related to the three glycyrrhizin-producing Glycyrrhiza spp. (G. uralensis, G. inflata, and G. glabra), even though G. bucharica does not produce glycyrrhizin.

Immunohistochemical analyses reveal FoxP3 expressions in spleen and colorectal cancer in mice treated with AOM/DSS, and their suppression by glycyrrhizin.

We previously demonstrated that glycyrrhizin (GL) suppressed inflammation and carcinogenesis in an azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced murine model of colorectal cancer (CC). In this study, we found an accumulation of regulatory T cells (Tregs) in the spleen and suppression by GL in model mice. ICR mice were divided into four groups: Control, GL, CC, and GL-treated CC (CC+GL), and were sacrificed 20 weeks after AOM/DSS treatment. We measured spleen weight, areas of white and red pulp, and CD8+ T cells (cytotoxic T lymphocytes, CTL), and CD11c-positive cells (dendritic cells) in splenic tissues and forkhead box protein 3 (FoxP3)-positive cells (Tregs) in colorectal and splenic tissues. In all cases, the CC group showed a significant increase compared with those in Control group, and GL administration significantly attenuated this increase. These results indicate that Tregs accumulated in the spleen may participate in inflammation-related carcinogenesis by suppressing CTL. We also suggest that GL which binds to high-mobility group box 1 (HMGB1), suppresses carcinogenesis with decreasing Tregs in the spleen. Furthermore, there was an expression of FoxP3 in cancer cells, indicating that it may be involved in the malignant transformation of cancer cells.

Glycyrrhizin and its derivatives promote hepatic differentiation via sweet receptor, Wnt, and Notch signaling.

The acute liver disease is involved in aberrant release of high-mobility group box 1 (HMGB1). Glycyrrhizin (GL), a traditional Chinese medicine for liver disease, binds to HMGB1, thereby inhibits tissue injury. However the mode of action of GL for chronic liver disease remains unclear. We investigated the effects of glycyrrhizin (GL) and its derivatives on liver differentiation using human iPS cells by using a flow cytometric analysis. GL promoted hepatic differentiation at the hepatoblast formation stage. The GL derivatives, 3-O-mono-glucuronyl 18ß-glycyrrhetinic acid (Mono) and 3-O-[glucosyl (1 â†’ 2)-glucuronyl] 18ß-glycyrrhetinic acid increased AFP+ cell counts and albumin+ cell counts. Glucuronate conjugation seemed to be a requirement for hepatic differentiation. Mono exhibited the most significant hepatic differentiation effect. We evaluated the effects of (±)-2-(2,4-dichlorophenoxy) propionic acid (DP), a T1R3 antagonist, and sucralose, a T1R3 agonist, on hepatic differentiation, and found that DP suppressed Mono-induced hepatic differentiation, while sucralose promoted hepatic differentiation. Thus, GL promoted hepatic differentiation via T1R3 signaling. In addition, Mono increased ß-catenin+ cell count and decreased Hes5+ cell count suggesting the involvement of Wnt and Notch signaling in GL-induced hepatic differentiation. In conclusion, GL exerted a hepatic differentiation effect via sweet receptor (T1R3), canonical Wnt, and Notch signaling.

Glycyrrhizin Derivatives Suppress Cancer Chemoresistance by Inhibiting Progesterone Receptor Membrane Component 1.

Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in various cancer cells and contributes to tumor progression. We have previously shown that PGRMC1 forms a unique heme-stacking functional dimer to enhance EGF receptor (EGFR) activity required for cancer proliferation and chemoresistance, and the dimer dissociates by carbon monoxide to attenuate its biological actions. Here, we determined that glycyrrhizin (GL), which is conventionally used to ameliorate inflammation, specifically binds to heme-dimerized PGRMC1. Binding analyses using isothermal titration calorimetry revealed that some GL derivatives, including its glucoside-derivative (GlucoGL), bind to PGRMC1 potently, whereas its aglycone, glycyrrhetinic acid (GA), does not bind. GL and GlucoGL inhibit the interaction between PGRMC1 and EGFR, thereby suppressing EGFR-mediated signaling required for cancer progression. GL and GlucoGL significantly enhanced EGFR inhibitor erlotinib- or cisplatin (CDDP)-induced cell death in human colon cancer HCT116 cells. In addition, GL derivatives suppressed the intracellular uptake of low-density lipoprotein (LDL) by inhibiting the interaction between PGRMC1 and the LDL receptor (LDLR). Effects on other pathways cannot be excluded. Treatment with GlucoGL and CDDP significantly suppressed tumor growth following xenograft transplantation in mice. Collectively, this study indicates that GL derivatives are novel inhibitors of PGRMC1 that suppress cancer progression, and our findings provide new insights for cancer treatment.

CJ-21,164, a new D-glucose-6-phosphate phosphohydrolase inhibitor produced by a fungus Chloridium sp.

A new D-glucose-6-phosphate phosphohydrolase (G6Pase) inhibitor, CJ-21,164 (1) was isolated from the fermentation broth of the fungus Chloridium sp. CL48903. The structure was elucidated to be a novel tetramer of the salicylic acid derivatives by spectroscopic analyses. Compound I inhibited G6Pase in rat liver microsomes with an IC50 of 1.6 microM. Glucose output from hepatocytes isolated from rat liver was inhibited when I was present in the incubation medium, consistent with the role of I as a G6Pase inhibitor.

Thielavins as glucose-6-phosphatase (G6Pase) inhibitors: producing strain, fermentation, isolation, structural elucidation and biological activities.

High-throughput screening of microbial extracts using rat hepatic microsomal glucose-6-phosphatase (G6Pase) led us to find thielavin B as a G6Pase inhibitor with inhibition of glucose output from glucagon-stimulated hepatocytes. Further searching for more potent analogs identified 11 new thielavins F-P in addition to the known thielavins A and B from a fungus Chaetomium carinthiacum ATCC 46463. Thielavin G showed the strongest activity as a G6Pase inhibitor (IC50=0.33 microM), while the IC50 of thielavin B was 5.5 microM. According to the structure-activity relationship, including authentic thielavins C, D and 3 partial hydrolysates from thielavins A and B, 3 benzoic acid-units and carboxylic acid functions are essential for G6Pase inhibition.

CJ-15,696 and its analogs, new furopyridine antibiotics from the fungus Cladobotryum varium: fermentation, isolation, structural elucidation, biotransformation and antibacterial activities.

CJ-15,696 and 7 novel furopyridine antibiotics were isolated from the fungus Cladobotryum varium CL12284. Their structures were determined by X-ray crystallography and spectral analysis. Three biotransformed analogs were also prepared from CJ-15,696. CJ-15,696 showed moderate activity against various Gram-positive bacteria including some drug resistant strains such as methicillin resistant Staphylococcus aureus (MRSA).

Physicochemical and structural properties of glycerin gel prepared using glycyrrhizic acid diethyl ester.

Glycyrrhizic acid diethyl ester (GZ-DE) was developed as a prodrug of glycyrrhizic acid (GZ), a hepatitis therapeutic drug. We fortuitously found that GZ-DE gels with glycerin selectively while searching for a safe solvent with which to dissolve GZ-DE. Based on this gelation, the aim of this study was to investigate the preparation of the gel and study the rheology, physicochemical and structural properties of the glycerin gel by differential scanning calorimeter (DSC), capillary electrophoresis (CEP), nuclear magnetic resonance (NMR), and small angle X-ray scattering (SAXS). The glycerin gel was prepared by the addition of at least 2.0% w/w GZ-DE. This gel did not flow at room temperature. After mixing glycerin and GZ-DE, a gel was formed after 2 days at 25°C or 3 h at 60°C. Glycerin gel containing 2.4% w/w GZ-DE provided the following results: 1) The glycerin gel exhibited creep at a constant stress of less than 10 Pa, but it is a fragile gel, showing Newtonian flow at 10 Pa stress. 2) Dynamic viscoelastic measurements showed that the elastic modulus (G') exceeds the viscous modulus (G''), indicating that glycerin gel has solid-like properties. 3) DSC showed a significant difference between the glass transition temperature of glycerin and glycerin gel. 4) CEP did not reveal a new compound in the glycerin gel. 5) NMR confirmed that glycerin gel is a physical gel. 6) SAXS measurements revealed that the glycerin gel has an oval-shaped basic frame (119 nm long and 65 nm wide).

Intestinal absorption and biliary elimination of glycyrrhizic acid diethyl ester in rats.

BACKGROUND: The purpose of this study was to evaluate absorption and elimination from the gastrointestinal tract of glycyrrhizic acid diethyl ester (GZ-DE) which was prepared as a prodrug of glycyrrhizic acid (a poorly absorbed compound) in rats. METHODS: After the GZ-DE solution was administered via the intravenous, intraduodenal, intraileal, and stomach routes, GZ-DE and GZ concentrations in bile were determined by high-performance liquid chromatography. The stability of GZ-DE was estimated from residual GZ-DE and GZ produced in GZ-DE solutions prepared with distilled water, a pH 1.2 solution, 0.9% NaCl solution, and phosphate-buffered solution (pH 7.4) at 37°C. RESULTS: GZ-DE was eliminated into bile by the pharmacokinetic parameters of apparent distribution rate constant (4.56 ± 0.36 per hour) and apparent elimination rate constant (0.245 ± 0.042 per hour). After intravenous and intraduodenal administration of GZ-DE, the concentration ratio of GZ-DE to GZ in bile was approximately 4:1, and the bioavailability of GZ containing GZ-DE was three-fold higher compared with the bioavailability of GZ after intraduodenal administration. GZ-DE was immediately precipitated in pH 1.2 solution and was converted to GZ by hydrolysis in pH 7.4 solution. CONCLUSION: Improvement of intestinal absorption of GZ was made possible by administration of GZ-DE into the intestine where absorption of GZ is lower than in the strong acidic environment of the stomach. However, because the elimination rate in bile simulated from kinetic parameters of GZ-DE was higher than the conversion rate from GZ-DE to GZ by hydrolysis, it is thought that the availability of GZ as a revolutionary prodrug was not high from the viewpoint of bioavailability of GZ in the liver by intestinal administration of GZ-DE.