Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. AIM OF THE STUDY: This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. MATERIALS AND METHODS: SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. RESULTS: Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a "closed" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. CONCLUSION: Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Extraction and Hypolipidemic Activity of Low Molecular Weight Polysaccharides Isolated from Rosa Laevigata Fruits.

Three novel low molecular weight polysaccharides (RLP-1a, RLP-2a, and RLP-3a) with 9004, 8761, and 7571 Da were first obtained by purifying the crude polysaccharides from the fruits of a traditional Chinese medicinal herb Rosae Laevigatae. The conditions for polysaccharides from the R. Laevigatae fruit (RLP) extraction were optimized by the response surface methodology, and the optimal conditions were as follows: extraction temperature, 93°C; extraction time, 2.8 h; water to raw material ratio, 22; extraction frequency, 3. Structural characterization showed that RLP-1a consisted of rhamnose, arabinose, xylose, glucose, and galactose with the ratio of 3.14 : 8.21 : 1 : 1.37 : 4.90, whereas RLP-2a was composed of rhamnose, mannose, glucose, and galactose with the ratio of 1.70 : 1 : 93.59 : 2.73, and RLP-3a was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose with the ratio of 6.04 : 26.51 : 2.05 : 1 : 3.17 : 31.77. The NMR analyses revealed that RLP-1a, RLP-2a, and RLP-3a contained 6, 4, and 6 types of glycosidic linkages, respectively. RLP-1a and RLP-3a exhibited distinct antioxidant abilities on the superoxide anions, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and hydroxyl radicals in vitro. RLPs could decrease the serum lipid levels, elevate the serum high-density lipoprotein cholesterol levels, enhance the antioxidant enzymes levels, and upregulate of FADS2, ACOX3, and SCD-1 which involved in the lipid metabolic processes and oxidative stress in the high-fat diet-induced rats. These results suggested that RLPs ameliorated the high-fat diet- (HFD-) induced lipid metabolism disturbance in the rat liver through the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Low molecular weight polysaccharides of RLP could be served as a novel potential functional food for improving hyperlipidemia and liver oxidative stress responses.

Physicochemical properties and digestion of the lotus seed starch-green tea polyphenol complex under ultrasound-microwave synergistic interaction.

Complex starch is gaining research attention due to its unique physicochemical and functional properties. Lotus seed starch (LS) suspensions (6.7%, w/v) with added green tea polyphenols (GTPs) (10%, w/w) were subjected to ultrasound (200-1000 W)-microwave (150-225 W) (UM) treatment for 15 min. The effects of UM treatment on the physicochemical properties of the LS-GTP system were investigated and exceeded that of microwave or ultrasound alone. The properties (morphology, X-ray diffraction pattern and so on) were affected by GTPs to various extents, depending on ultrasonic power. These influences may be explained by the non-covalent interactions between GTPs and LS. V-type LS-GTP inclusion complex and non-inclusive complex formation were observed. Their morphology and the distribution of GTPs molecules within them were estimated using scanning electron microscopy and confocal laser scanning microscopy. Furthermore, the digestion of LS-GTP complex was investigated by a dynamic in vitro rat stomach-duodenum (DIVRSD) model, lower digestion efficiency of LS has been achieved and the residues showed gradual improvement in morphology. These all experimental results do provide new insight into the complex starch production.

Lotus seed skin proanthocyanidin extract exhibits potent antioxidant property via activation of the Nrf2-ARE pathway.

Lotus seed is well known as traditional food and medicine, but its skin is usually discarded. Recent studies have shown that lotus seed skin contains a high concentration of proanthocyanidins that have multi-functions, such as antioxidation, anti-inflammation, and anti-cancer effects. In the present study, we aimed to isolate and purify the proanthocyanidins from lotus seed skin by acetone extraction and rotary evaporation, identify their chemical structures by HPLC-MS-MS and NMR, and further investigate the antioxidant properties of the extract purified by macroporous resin (PMR) from lotus seed skin both in vitro and in vivo. The results showed that PMR mainly contained oligomeric proanthocyanidins, especially dimeric procyanidin B1 (PB1), procyanidin B2 and procyanidin B4. Although it had limited ability to directly scavenge radicals in vitro, PMR could significantly enhance the expressions of antioxidant proteins via activation of nuclear factor-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway in HepG2 cells. Molecular data revealed that PB1, a major component in PMR, stabilized Nrf2 by inhibiting the ubiquitination of Nrf2, which led to subsequent activation of the Nrf2-ARE pathway, including the enhancements of Nrf2 nuclear translocation, Nrf2-ARE binding and ARE transcriptional activity. Moreover, the in vivo results in high fat diet-induced mice further verified the powerful antioxidant property of PMR. These results revealed that lotus seed skin is a promising resource for functional food development.

[Comparative analysis on seedling growth dynamics and physiological indices of Bupleurum smithii var. parvifolium and B. scorzonerifolium].

With Bupleurum smithii var. parvifolium and B. scorzonerifolium as test objects, in order to provide a theoretical basis for the introduction and domestication of B. smithii var. parvifolium, the growth and development dynamics of seedlings, biomass accumulation, the content of malonaldehyde(MDA), the activity of antioxidase such as SOD, POD, CAT and APX between them were comparatively analyzed by direct sowing culture in the open field. The results indicated that the morphological index and the biomass accumulation of B. smithii var. parvifolium such as root diameter, root length, plant height and leaf number were inferior to B. scorzonerifolium, the antioxidase SOD and POD activity of B. smithii var. parvifolium was significantly inferior to B. scorzonerifolium (P<0.05), the antioxidase CAT and APX activity of B. smithii var. parvifolium was inferior to B. scorzonerifolium but the difference wasn't significant, while MDA content was superior to B. scorzonerifolium(P<0.05). Thus, compared with cultivated B. scorzoneri folium, the plant growth velocity of wild B. smithii var. parvifolium was relatively slower and its resistance was relatively weaker after introduction and domestication.

Baicalein modulates Nrf2/Keap1 system in both Keap1-dependent and Keap1-independent mechanisms.

Baicalein, a major component of Scutellaria baicalensis Georgi (Huang Qin), is widely used in the traditional Chinese medicine. However, the mechanisms underlying cancer chemoprevention are still not clear. The present study aimed to clarify how baicalein modulate Nrf2/Keap1 system to exert its cytoprotection and cancer chemoprevention. In the upstream cellular signaling, baicalein stimulated the phosphorylation of MEK1/2, AKT and JNK1/2, which were demonstrated to be essential for baicalein-induced Nrf2 expression by their inhibitors. Immunoprecipitation with Nrf2 found that baicalein increased the amount of phosphorylated MEK1/2, AKT and JNK1/2 bound to Nrf2, and also stabilized Nrf2 protein by inhibiting the ubiquitination and proteasomal turnover of Nrf2. Simultaneously, baicalein down-regulated Keap1 by stimulating modification and degradation of Keap1 without affecting the dissociation of Keap1-Nrf2. Silencing Nrf2 using Nrf2 siRNA markedly reduced the ARE activity under both baseline and baicalein-induced conditions. Thus, baicalein positively modulate Nrf2/Keap1 system through both Keap1-independent and -dependent pathways. These finding provide an insight to understand the mechanisms of baicalein in cytoprotection and cancer chemoprevention.

Profiling and characterisation by liquid chromatography/multi-stage mass spectrometry of the chlorogenic acids in Gardeniae Fructus.

The chlorogenic acids of Gardeniae Fructus used traditionally as a Chinese herbal medicine (zhizi) have been investigated qualitatively by liquid chromatography/multi-stage mass spectrometry (LC/MS(4)). Twenty-nine chlorogenic acids were detected and twenty-five characterised to regioisomer level on the basis of their fragmentation, twenty-four for the first time from this source. Assignment to the level of individual regioisomers was possible for three caffeoylquinic acids, three dicaffeoylquinic acids, three sinapoylquinic acids, four caffeoyl-sinapoylquinic acids, two feruloyl-sinapoylquinic acids, one p-coumaroyl-sinapoylquinic acid, three (3-hydroxy, 3-methyl)glutaroylquinic acids, two (3-hydroxy, 3-methyl)glutaroyl-feruloylquinic acids, one (3-hydroxy, 3-methyl)glutaroyl-dicaffeoylquinic acid, and one (3-hydroxy, 3-methyl)glutaroyl-caffeoyl-feruloylquinic acid. Six (3-hydroxy, 3-methyl)glutaroyl-caffeoylquinic acids were detected and two were tentatively assigned as 3-caffeoyl-4-(3-hydroxy, 3-methyl)glutaroylquinic acid and 3-caffeoyl-5-(3-hydroxy, 3-methyl)glutaroylquinic acid. The (3-hydroxy, 3-methyl)glutaroyl residue modifies the mass spectral fragmentation behavior and elution sequence compared with the chlorogenic acids that contain only a cinnamic acid residue(s). Fourteen of these twenty-nine chlorogenic acids have not previously been reported from any source.

Profiling the chlorogenic acids and other caffeic acid derivatives of herbal chrysanthemum by LC-MSn.

Four samples of herbal chrysanthemum have been profiled qualitatively by LC-MS5 to identify their component chlorogenic acids and partially characterize other caffeic acid derivatives. The chlorogenic acids were minor components, and the four samples varied markedly in profile. Three p-coumaroylquinic acids, three feruloylquinic acids, four caffeoylquinic acids, six dicaffeoylquinic acids, and two tricaffeoylquinic acids were detected, 13 for the first time from this source. Partial characterization of minor components suggested the presence of five caffeoyl-hexose esters and caffeic acid-4-beta-d-glucose that have not previously been reported from this source, and eight caffeoylquinic acid glycosides and 16 dicaffeoylquinic acid glycosides that have not previously been reported in nature. Succinic acid-containing chlorogenic acids and chlorogenic acids based on epi-quinic acid, previously reported in Chrysanthemum spp., were not detected in these samples.

[Responses of litter decomposition and nutrient release to simulated nitrogen deposition in an evergreen broad-leaved forest in southwestern Sichuan].

An in situ experiment was conducted in an evergreen broad-leaved forest in southwestern Sichuan to study the responses of litter decomposition and nutrient release to simulated nitrogen deposition. Four treatments were installed, i.e., null level (CK), low nitrogen level (LN, 50 kg N x hm(-2) x a(-1)), moderate nitrogen level (MN, 100 kg N x hm(-2) x a(-1)) and high nitrogen level (HN, 150 kg N x hm(-2) x a(-1)). The results showed that it would take 4.72-6.33 years to decompose 95% of litter mass, with the highest decomposition rate in CK and the lowest one in HN. After 365 days, the litter decomposition rate in N-amended treatments was lower than that in CK, but significant difference was only observed between HN and CK (P < 0.05). The remained C was higher, and the remained N and K were significantly higher in N-amended treatments than in CK (P < 0.05). The remained P was also higher in N-amended treatments than in CK, but significant difference was only observed between LN and CK (P < 0.05). Compared with CK, all N-amended treatments had a 3.9%-23.7% increase of litter C/N ratio. During litter decomposition, element N featured a pattern of accumulation at early stage and release later, while C, P and K released all the time. Nitrogen deposition inhibited both the nutrient release from the litter and the decomposition of its lignin and cellulose. The effects of nitrogen deposition on litter decomposition in the forest changed from positive to negative as time passed, and the negative effect could be strengthened with the increase of deposited nitrogen concentration.

Hypermethylation of the death-associated protein kinase promoter attenuates the sensitivity to TRAIL-induced apoptosis in human non-small cell lung cancer cells.

Death-associated protein (DAP) kinase plays an important role in IFN-gamma, tumor necrosis factor (TNF)-alpha, or Fas-ligand induced apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand family and can induce caspase-dependent apoptosis in cancer cells while sparing most of the normal cells. However, some of the cancer cell lines are insensitive to TRAIL, and such resistance cannot be explained by the dysfunction of TRAIL receptors or their known downstream targets. We reported previously that DAP kinase promoter is frequently methylated in non-small cell lung cancer (NSCLC), and such methylation is associated with a poor clinical outcome. To determine whether DAP kinase promoter methylation contributes to TRAIL resistance in NSCLC cells, we measured DAP kinase promoter methylation and its gene expression status in 11 NSCLC cell lines and correlated the methylation/expression status with the sensitivity of cells to TRAIL. Of the 11 cell lines, 1 had a completely methylated DAP kinase promoter and no detectable DAP kinase expression, 4 exhibited partial promoter methylation and substantially decreased gene expression, and the other 6 cell lines showed no methylation in the promoter and normal DAP kinase expression. Therefore, the amount of DAP kinase expression amount was negatively correlated to its promoter methylation (r = -0.77; P = 0.003). Interestingly, the cell lines without the DAP kinase promoter methylation underwent substantial apoptosis even in the low doses of TRAIL, whereas those with DAP kinase promoter methylation were resistant to the treatment. The resistance to TRAIL was reciprocally correlated to DAP kinase expression in 10 of the 11 cell lines at 10 ng/mL concentration (r = 0.91; P = 0.001). We treated cells resistant to TRAIL with 5-aza-2'-deoxycytidine, a demethylating reagent, and found that these cells expressed DAP kinase and became sensitive to TRAIL. These results suggest that DAP kinase is involved in TRAIL-mediated cell apoptosis and that a demethylating agent may have a role in enhancing TRAIL-mediated apoptosis in some NSCLC cells by reactivation of DAP kinase.

Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry.

Despite improvements in treatment of patients with head and neck squamous cell carcinoma (HNSCC) over the last two decades, the survival rate of these patients has not increased significantly. One of the major factors in the poor outcome of the disease is regional metastasis. To better understand the mechanisms of this process at the protein level, we performed two-dimensional electrophoresis (2-DE) and mass spectrometry using SELDI ProteinChip technology to identify proteins differentially expressed in two HNSCC cell lines, UMSCC10A and UMSCC10B, from the same patient. UMSCC10A was derived from the primary tumor and UMSCC10B from a metastatic lymph node. The differentially expressed proteins were excised from the gels. Following in-gel digestion by trypsin, mass profiles of the peptides were generated. Proteins were identified by submitting the peptide mass profiles to a public available NCBInr databases (www.proteometrics.com). Two membrane-associated proteins, annexin I and annexin II and glycolytic protein enolase-alpha were found to be upregulated, and calumenin precursor down-regulated, in metastatic cell line UMSCC10B. The identity of these proteins was confirmed by analyzing additional peptide mass fingerprints obtained by endoproteinase lysine-C digestion. The results were also validated by Western blotting analysis. Our results showed that enolase-alpha, annexin-I and annexin-II might be important molecules in head and neck cancer invasion and metastasis. The results also suggest an important complementary role for proteomics in identification of molecular abnormalities important in cancer development and progression.