Linderapyrone analogue LPD-01 as a cancer treatment agent by targeting importin7.

The Wnt/ß-catenin signaling pathway plays important roles in several cancer cells, including cell proliferation and development. We previously succeeded in synthesizing a small molecule compound inhibiting the Wnt/ß-catenin signaling pathway, named LPD-01 (1), and 1 inhibited the growth of human colorectal cancer (HT-29) cells. In this study, we revealed that 1 inhibits the growth of HT-29 cells stronger than that of another human colorectal cancer (SW480) cells. Therefore, we have attempted to identify the target proteins of 1 in HT-29 cells. Firstly, we investigated the effect on the expression levels of the Wnt/ß-catenin signaling pathway-related proteins. As a result, 1 inhibited the expression of target proteins of Wnt/ß-catenin signaling pathway (c-Myc and Survivin) and their genes, whereas the amount of transcriptional co-activator (ß-catenin) was not decreased, suggesting that 1 inhibited the Wnt/ß-catenin signaling pathway without affecting ß-catenin. Next, we investigated the target proteins of 1 using magnetic FG beads. Chemical pull-down assay combined with mass spectrometry suggested that 1 directly binds to importin7. As expected, 1 inhibited the nuclear translocation of importin7 cargoes such as Smad2 and Smad3 in TGF-ß-stimulated HT-29 cells. In addition, the knockdown of importin7 by siRNA reduced the expression of target genes of Wnt/ß-catenin signaling pathway. These results suggest that importin7 is one of the target proteins of 1 for inhibition of the Wnt/ß-catenin signaling pathway.

Highly sensitive indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody against saikosaponin b2 for quality control of Kampo medicines containing Bupleuri radix.

Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix-containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95-125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph-mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix.

Cytotoxic activities of alkaloid constituents from the climbing stems and rhizomes of Sinomenium acutum against cancer stem cells.

From the methanolic extract of the climbing stems and rhizomes of Sinomenium acutum, two new aporphine analogues, acutumalkaloids I and II, were isolated together with fifteen known compounds including lysicamine. The chemical structures of the isolated new compounds were elucidated based on chemical/physicochemical evidence such as NMR and MS spectra. For acutumalkaloids I and II, the absolute configurations were established by comparison of experimental and predicted electronic circular dichroism (ECD) data. We compared anti-proliferative activities of isolated compounds with reported naturally occurring Wnt/ß-catenin pathway inhibitor, nuciferine. Among the isolated compounds, we found lysicamine have anti-proliferative activity against both of HT-29 human colon cancer cell line and its cancer stem cells (CSCs). The IC50 values of lysicamine against non-CSCs and its CSCs were lower than that of nuciferine. In addition, the results of western blotting analysis suggested that lysicamine inhibited the expression of Wnt/ß-catenin pathway target protein such as survivin. These results suggested that lysicamine show cytotoxic activity via inhibition of Wnt/ß-catenin pathway.

Chemical Structures and Anti-proliferative Effects of Valeriana fauriei Constituents on Cancer Stem Cells.

We isolated the new sesquiterpenes, valerianaterpenes IV and V, and the new lignans valerianalignans I-III from the methanol extracts of the rhizomes and roots of Valeriana fauriei and elucidated their structures based on chemical and spectroscopic findings. The absolute configuration of valerianaterpene IV and valerianalignans I-III were established by comparing experimental and predicted electronic circular dichroism (ECD) data. Among the isolated compounds, valerianalignans I and II exerted anti-proliferative activity against human astrocytoma cells (U-251 MG) and their cancer stem cells (U-251 MG CSCs). Interestingly, valerianalignans I and II notably exerted anti-proliferative activities at lower concentrations against CSCs than non-CSCs, and the absolute configurations of these compounds affected their activities.

Angiotensin I-Converting Enzyme-Inhibitory Activity and Phytochemical Profile of Constituents of the Leaves of Rehmannia glutinosa f. hueichingensis.

The root of Rehmannia glutinosa Liboschitz forma hueichingensis HSIAO has been used as a tonic and treatment for urinary and skin disorders in Japanese Kampo medicine. Phytochemical investigation of the root has been well reported, but that of the leaves is limited. To explore the potential value of R. glutinosa leaves, we focused on the angiotensin I-converting enzyme (ACE)-inhibitory activity. The leaf extract exhibited ACE-inhibitory activity, and the inhibitory potency of leaves was stronger than that of roots. Using this activity as an indicator, we isolated linaride (1), 6-O-hydroxybenzoyl ajugol (2), acteoside (3), leucosceptoside A (4), martynoside (5), luteolin (6), apigenin (7), and chrysoeriol (8) by separating and purifying the extract. We then examined the ACE-inhibitory activities of 1-8, catalpol (9), aucubin (10), ajugol (11), and echinacoside (12). Among them, 3, 6, and 12 displayed the most potent inhibitory activity. A simultaneous analytical method was also developed using compounds contained in R. glutinosa leaves and roots, and their contents were compared. The method consisted of extraction with 50% aqueous methanol under sonication for 60 min and LC/MS measurement. R. glutinosa leaves tended to have higher levels of majority of the analytes than the roots, including 3 and 6, which had higher ACE-inhibitory activity. These results suggest that 3 and 6 contribute to the ACE-inhibitory activity of R. glutinosa leaves, which may represent a useful medicinal resource for hypertension.

Iridoids from Morinda lucida, (Benth.) Rubiaceae, produced analgesic and anti-inflammatory activities via agonism at the kappa and delta opioid receptors, inhibition of COX-2 besides elevation of CAT and SOD activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Pain and inflammation are the major symptoms of almost every human disease. Herbal preparations from Morinda lucida are used to treat pain and inflammation in traditional medicine. However, the analgesic and anti-inflammatory activities of some of the plant's chemical constituents are not known. AIM OF THE STUDY: The aim of this study is to evaluate the analgesic and anti-inflammatory activities and possible mechanisms of these activities of iridoids from Morinda lucida. MATERIAL AND METHODS: The compounds were isolated using column chromatography and characterized by NMR spectroscopy and LC-MS. Anti-inflammatory activity was evaluated using carrageenan-induced paw edema. Whereas, the analgesic activity was assessed in the hot plate and acetic acid-induced writhing assays. Mechanistic studies were conducted using pharmacological blockers, determination of antioxidant enzymes, lipid peroxidation, and docking studies. RESULTS: The iridoid, ML2-2 exhibited inverse dose-dependent anti-inflammatory activity (42.62% maximum at 2 mg/kg p. o). ML2-3 produced dose-dependent anti-inflammatory activity (64.52% maximum at 10 mg/kg p. o.). Anti-inflammatory activity of diclofenac sodium was 58.60% at 10 mg/kg p. o. Furthermore, ML2-2 and ML2-3 produced analgesic activity (P < 0.01) of 44.44 ± 5.84 and 54.18 ± 19.01%. at 10 mg/kg p. o. respectively in the hot plate assay and 64.88 and 67.44% in the writhing assay. ML2-2 significantly elevated catalase activity. However, ML2-3 elevated SOD and catalase activity significantly. In the docking studies, both iridoids formed stable crystal complexes with delta and kappa opioid receptors, and the COX-2 enzyme with very low free binding energies (ΔG) from -11.2 to -14.0 kcal/mol. However, they did not bind with the mu opioid receptor. The lower bound RMSD of most of the poses were found to be ≤ 2. Several amino acids were involved in the interactions through various inter molecular forces. CONCLUSION: These results indicate that ML2-2 and ML2-3 possessed very significant analgesic and anti-inflammatory activities via acting as both delta and kappa opioid receptor agonist, elevation of anti-oxidant activity and inhibition of COX-2.

Anti-Proliferative Effects of Iridoids from Valeriana fauriei on Cancer Stem Cells.

We isolated seven new iridoid glucosides (valerianairidoids I-VII; 1-3, 6, 7, 9, and 12) and six known compounds from the methanol extract of the dried rhizomes and roots of Valeriana fauriei. Chemical and spectroscopic data were used to elucidate the chemical structures of the seven new iridoid glucosides, and their absolute configurations were determined by comparing their electronic circular dichroism (ECD) spectra with those determined experimentally. Aglycones 1a, 6a, and 9a, which were obtained by enzymatic hydrolysis of the isolated iridoid glucosides, exhibited anti-proliferative activities against cancer stem cells (CSCs) established by a sphere-formation assay using human breast cancer (MDA-MB-231) and human astrocytoma (U-251MG) cells. Interestingly, these iridoids selectively showed anti-proliferative activities against CSCs from MDA-MB-231 cells. These results suggest that the iridoids obtained in this study may have potency as a breast cancer treatment and as preventive agent via exterminating CSCs.

Bioassay-guided Fractionation of Clove Buds Extract Identifies Eugenol as Potent Melanogenic Inducer in Melanoma Cells.

Clove, a dried flower buds of Syzygium aromaticum, is used in traditional medicine, for culinary purposes, and in essential oil production. In our preliminary screening of crude drugs used in Japanese Kampo formulas, a methanol (MeOH) extract of clove buds was found to exhibit a melanin induction. To date, the effects of clove buds or their constituents on the activation of melanogenesis remain unclear. Thus, this study aimed to isolate active compounds from the MeOH extract of clove buds associated with melanin synthesis in melanoma cells and to investigate the molecular mechanism involved. The MeOH extract of clove buds increased melanin content in murine B16-F1 melanoma cells. To identify the active compounds responsible for melanin induction, the MeOH extract was suspended in water and successively partitioned using hexane, ethyl acetate (EtOAc), and n-butanol (n-BuOH). Comparative analysis revealed that the EtOAc fraction induced melanin synthesis. Bioassay-guided separation of the EtOAc fraction isolated three compounds including eugenol. The analysis of structure-activity relationships of eugenol and structurally related compounds indicated that eugenol was the most potent melanin inducer among the 11 compounds, and that a hydroxyl group at C-1 and a methoxy group at C-2 may contribute to melanin induction. Eugenol induced melanin synthesis in human HMV-II melanoma cells as well as in B16-F1 cells. Further analysis indicated that eugenol may invoke intracellular tyrosinase activity and expression of tyrosinase, tyrosinaserelated protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF). These results suggest that eugenol enhances melanin synthesis by upregulating the expression of MITF and subsequent expression of melanogenic enzymes, and that it may be a potent therapeutic agent for hypopigmentation.

(+)-Magnolin Enhances Melanogenesis in Melanoma Cells and Three-Dimensional Human Skin Equivalent; Involvement of PKA and p38 MAPK Signaling Pathways.

Magnoliae Flos is a traditional herbal medicine used to treat nasal congestion associated with headache, empyema, and allergic rhinitis. In our preliminary screening of crude drugs used in Japanese Kampo formulas for melanin synthesis, the methanol extract of Magnoliae Flos was found to exhibit strong melanin synthesis activity. However, there have been no studies evaluating the effects of Magnoliae Flos or its constituents on melanogenesis. The present study aimed to isolate the active compounds from Magnoliae Flos that activate melanin synthesis in melanoma cells and three-dimensional human skin equivalent, and to investigate the molecular mechanism underlying melanin induction. The methanol extract of Magnoliae Flos induced an increase of melanin content in both B16-F1 and HMV-II cells. A comparison of melanin induction by three fractions prepared from the extract showed that the ethyl acetate fraction markedly induced melanin synthesis. Bioassay-guided separation of the ethyl acetate fraction resulted in the isolation of seven lignans (1:  - 7: ). Among them, (+)-magnolin (5: ) strongly induced melanin synthesis and intracellular tyrosinase activity. Furthermore, the ethyl acetate fraction and 5: clearly induced melanin content in a three-dimensional human skin equivalent. Molecular analysis revealed that 5: triggered the protein expression of tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. Further analysis of transcriptional factors and signaling pathways demonstrated that 5: induces the protein expression of tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2 activated by the protein kinase A- and p38 mitogen-activated protein kinase-dependent pathways, leading to cAMP-responsive element-binding protein phosphorylation and microphthalmia-associated transcription factor expression. These findings demonstrate the potential of 5: as a potent therapeutic agent for hypopigmentation.

Chemical structures and induction of cell death via heat shock protein inhibition of the prenylated phloroglucinol derivatives isolated from Hypericum erectum.

Four new prenylated phloroglucinol derivatives (+)-erectumol I (1a), (-)-erectumol I (1b), (-)-erectumol II (2a), and (+)-erectumol II (2b) were isolated from the methanol extracts of the whole plants of Hypericum erectum. These new compounds were isolated as a pair of enantiomers, respectively. The planar chemical structures and relative configurations of the new compounds were suggested by Cu-Kα X-ray diffraction analysis and been confirmed by high-resolution mass and 1D and 2D NMR spectroscopic data. The absolute configuration of the four new compounds were established by comparing the experimental and predicted electronic circular dichroism data. Isolated compounds 1b and 2b induced death of Adriamycin-treated HeLa cells. Their enantiomers 1a and 2a did not. In addition, the apparent mechanism of cell death of 1b was the inhibited expression of heat shock protein 105.

Construction of sulfur-containing compounds with anti-cancer stem cell activity using thioacrolein derived from garlic based on nature-inspired scaffolds.

Sulfur-containing compounds, such as cyclic compounds with a vinyl sulfane structure, exhibit a wide range of biological activities including anticancer activity. Therefore, the development of efficient strategies to synthesize such compounds is a remarkable achievement. We have developed a unique approach for the rapid and modular preparation of nature-inspired cyclic and acyclic sulfur-containing compounds using thioacrolein, a naturally occurring chemically unstable intermediate. We constructed thiopyranone derivatives through the regioselective sequential double Diels-Alder reaction of thioacrolein produced by allicin, a major component in garlic, and two molecules of silyl enol ether as the diene partner. The cytotoxicity toward cancer stem cells of the thiopyranones was equal to or higher than that of (Z)-ajoene (positive control) derived from garlic, and the thiopyranones had higher chemical stability than (Z)-ajoene.

Cell death-inducing activities via P-glycoprotein inhibition of the constituents isolated from fruits of Nandina domestica.

Two new pyrrole alkaloids methyl-E-mangolamide (1) and methyl-Z-mangolamide (2), four new megastigmane glycosides nandinamegastigmanes I-IV (3-6), and eight known compounds (7-14) were isolated from the methanol extract of the fruits of Nandina domestica. The structures of the new compounds were elucidated based on chemical and spectroscopic evidence. The absolute stereochemistry of nandinamegastigmane I (3) was established upon comparing the experimental and predicted electronic circular dichroism (ECD) data. Among the isolated compounds, 1 and 2 showed cell death-inducing activity on the Adriamycin-treated HeLa cells. In addition, one of the mechanisms for cell death-inducing activity of 1 and 2 was suggested as inhibition of P-glycoprotein.

Limonoids isolated from the Fortunella crassifolia and the Citrus junos with their cell death-inducing activity on Adriamycin-treated cancer cell.

From the fruits of Fortunella crassifolia and the peels of Citrus junos, two new limonoids, fortunellone and junosol were isolated together with three known compounds including nomilin. The chemical structures of the new compounds were elucidated based on chemical/physicochemical evidence. For fortunellone, the absolute configuration was established by comparison of experimental and predicted electronic circular dichroism (ECD) data. Fortunellon and nomilin significantly increased the number of dead cells on adriamycin (ADR)-treated human cervical cancer cells (HeLa). On the other hand, fortunellon and nomilin did not affects the number of dead cells alone. These results suggested that fortunellone and nomilin may have the potency as the chemotherapy enhancement agents.

Costunolide and dehydrocostuslactone from Saussurea lappa root inhibit autophagy in hepatocellular carcinoma cells.

Autophagy is a catabolic process for degradation of intracellular components and plays an important role in the development and growth of cancer. Our preliminary screening confirmed that an extract from the root of Saussurea lappa remarkably suppressed the proliferation of HepG2 hepatocellular carcinoma cells and inhibited autophagy. In this study, we explored the effects of costunolide (CL) and dehydrocostuslactone (DCL), which are bioactive sesquiterpene lactones in this extract, on autophagy modulation in HepG2 cells. An analysis of autophagy-related proteins demonstrated that CL and DCL blocks the autophagy process that leads to the accumulation of microtubule-associated protein 1 light chain 3 (LC3) and SQSTM1/p62 (p62). LC3 turnover assays indicated that CL and DCL trigger autophagy inhibition by blocking the autophagic flux, thereby resulting in the accumulation of LC3-II and p62. These results are encouraging and warrant further study of CL and DCL for potential use as autophagy inhibiting agents for liver cancer therapy.

Cytotoxic activities of sesquiterpenoids from the aerial parts of Petasites japonicus against cancer stem cells.

From the methanolic extract of the aerial parts of Petasites japonicus, six new eremophilane-type sesquiterpenoids, petasitesterpenes I-VI were isolated together with eight known compounds including S-japonin and eremophilenolide. The chemical structures of the isolated new compounds were elucidated based on chemical/physicochemical evidence. For petasitesterpenes I and II, the absolute configurations were established by comparison of experimental and predicted electronic circular dichroism (ECD) data. Among the isolated compounds, petasitesterpenes I, II, VI, and S-japonin showed cytotoxic activity against both human astrocytoma U-251MG cancer cells (non-CSCs) and their cancer stem cells (CSCs) isolated by sphere formation. In addition, cytotoxic activities of these compounds against breast cancer MDA-MB-231 were evaluated, supporting that petasitesterpene II has more effective than other isolated compounds.

Arctigenin suppresses cell proliferation via autophagy inhibition in hepatocellular carcinoma cells.

Autophagy is a catabolic process that degrades dysfunctional proteins and organelles and plays critical roles in cancer development. Our preliminary screening identified that extracts of the fruits of Arctium lappa and the fruits of Forsythia suspensa notably suppressed the proliferation of hepatocellular carcinoma HepG2 cells and downregulated the autophagy. In this study, we explored the effect of arctigenin (ARG), a bioactive lignan in both extracts, on cell proliferation and autophagy-related proteins in HepG2 cells. ARG inhibited the proliferation of HepG2 cells. Analysis of autophagy-related proteins demonstrated that ARG might block the autophagy that leads to sequestosome 1/p62 (p62) accumulation. The stage of inhibition in autophagy by ARG differed from those by the autophagy inhibitors 3-methyladenine (3-MA) or chloroquine (CQ). ARG could also inhibit starvation-induced autophagy. Further analysis of apoptosis-related proteins indicated that ARG did not affect caspase-3 activation and PARP cleavage, suggesting that the antiproliferative effect of ARG can occur independently of apoptosis. In summary, our study showed that ARG suppresses cell proliferation and inhibits autophagy, and might lead to the development of agents for autophagy research and cancer chemoprevention.

Chemical structures and cytotoxic activities of the constituents isolated from Hibiscus tiliaceus.

Five new cadinene-type sesquiterpenes, hibiscusterpenes I-V (1-5), and six known compounds (6-11) have been isolated from the methanol extract of the stems and the twigs of Hibiscus tiliaceus. The structures of the new compounds were elucidated based on chemical and spectroscopic evidence. The absolute stereochemistry of hibiscusterpene I (1) was determined using X-ray crystallography. For hibiscusterpene III (3), the absolute configuration was established upon comparison of the experimental and predicted electronic circular dichroism (ECD) data. Among the isolated compounds, hibiscone C (7) and syriacusin A (11) showed cytotoxic activity in HeLa cells. In addition, their cell death-inducing activity was observed using time-lapse cell imaging.

The old pharmaceutical oleoresin labdanum of Cistus creticus L. exerts pronounced in vitro anti-dengue virus activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The haemorrhagic dengue fever affects up to 500 million patients, annually causing 20.000 deaths, with no chemotherapeutic agent available. The oleoresin labdanum of Cistus creticus L. has been established as an anti-infective agent since antiquity in Mediterranean ethnopharmacology. MATERIALS AND METHODS: We tested several extracts and fractions of labdanum - standardised on labdane-type diterpenes via GC-MS - on their activity against the dengue virus (DENV-2 strain 00st-22A) in in vitro Vero cell cultures (96-well-plates, 5 days). Preliminary experiments with a labdanum diethyl ether raw-extract did not yield measureable results due to cytotoxic effects against Vero cells. In all following experiments, cell viability was constantly checked using the MTT-test. Fractionation of this raw-extract by liquid-liquid-extraction and column-chromatography on silica-gel (gradient elution with hexane, EtOAc, CHCl3, MeOH) succeeded in separating the anti-viral activity of labdanum from its cytotoxic effect. RESULTS: In the most active fraction GS5 at 30 µg/ml, dengue virus proliferation was 100% suppressed and cell viability was over 90%. Structural elucidation of major constituents of GS5 is currently ongoing, but thin-layer chromatography showed that this fraction is mainly dominated by manoyloxides, a class of labdane-type diterpenes with known antimicrobial activity. Claims concerning the antiviral activity of above ground parts of C. creticus have been made previously, but these generally ascribe this activity to hot water soluble polyphenols and propose an unspecific tanning effect of the viral surface proteins as the mechanism of action. However, the water soluble fraction enhanced viral proliferation. CONCLUSION: We therefore describe a direct, pharmacological, antiviral activity of a diethyl ether extract of labdanum against a virulent haemorrhagic fever like dengue for the first time.

Screening of Crude Drugs Used in Japanese Kampo Formulas for Autophagy-Mediated Cell Survival of the Human Hepatocellular Carcinoma Cell Line.

Background: Autophagy is a catabolic process through which dysfunctional proteins and organelles are degraded, and that is associated with the proliferation of cancer cells. The aim of this study was to screen approximately 130 kinds of crude drugs used in Japanese Kampo formulas to identify crude drugs that would regulate the proliferation through autophagy of human hepatocellular carcinoma HepG2 cells. Methods: Extracts of each crude drug were prepared using methanol. Protein levels were determined using Western blotting. Cell viability was measured by MTT assay. Results: Among the 130 crude extracts, 24 of them increased LC3-II expression. Among these, Goboshi (burdock fruit), Soboku (sappan wood), Mokko (saussurea root), Rengyo (forsythia fruit), and Hikai (dioscorea) notably suppressed the proliferation of HepG2 cells and increased p62 expression levels, which suggested that these five extracts downregulate the autophagic activity resulting in the accumulation of p62. On the other hand, Hishinomi (water chestnut), Biwayo (loquat leaf), and Binroji (areca) induced cell growth and decreased or were uninvolved with p62 expression levels, which implied that these three extracts might induce autophagy modulators for cell growth. Conclusions: The results suggest that the compounds contained in the crude drugs selected for this study could control cell viability by regulating autophagic activity in HepG2 cells. The isolation and identification of the active compounds in these drugs might lead to the development of agents for autophagy research and cancer chemoprevention.

Structures of triterpenoids from the leaves of Lansium domesticum.

From the methanolic extract of the leaves of Lansium domesticum, three new onoceranoid-type triterpenoids, lansium acids X-XII and a new cycloartane-type triterpene, lansium acid XIII, were isolated. The chemical structures of the isolated new compounds were elucidated on the basis of chemical/physicochemical evidence. For new onoceranoid-type triterpenoids, the absolute configurations were established by comparison of experimental and predicted electronic circular dichroism (ECD) data. The isolated onoceranoid-type triterpenoids showed antimutagenic effects in the Ames assay against 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1).