In vitro selection of aptamer S1 against MCF-7 human breast cancer cells.

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (Kd) value of 29.9 ±â€¯6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.

Diarylpentanol constituents from the aerial part of Stelleropsis tianschanica.

Five diarylpentanol derivatives including two new compounds stellerasme A (1), stellerasme B (2) were isolated from the aerial parts of Stelleropsis tianschanica. Their structures were elucidated by various spectroscopic techniques (UV, IR, MS, CD, 1D and 2D NMR). All compounds were evaluated for their cytotoxicity activity against HeLa and KB cell lines, and compound 1 showed selective activities against HeLa cell line with an IC50 value of 7.4 µM.

Tissue distribution of emulsified γ-tocotrienol and its long-term biological effects after subcutaneous administration.

BACKGROUND: γ-tocotrienol (GT3), an analogue of vitamin E, has gained increasing scientific interest recently as it provides significant health benefits. It has been shown that emulsified GT3, after subcutaneous administration, has long-term biological effects. However, whether the effects are due to the increase of GT3 level in the early phase following administration or the persistent functions after accumulation in tissues is unknown. This study was conducted to determine the levels of GT3 in different tissues by high performance liquid chromatography (HPLC) with a fluorescence detector after a single-dose of GT3 with polyethylene glycol (PEG-400) emulsion via subcutaneous injection. Previous studies have explored that GT3 has favorable effects on bone and can inhibit osteoclast formation. To confirm the persistent biological activity of accumulated GT3 in tissues, receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) gene expressions, which have an important role in regulating osteoclast formation, were also evaluated in bone tissue on day 1, 3, 7 and 14 after a signal subcutaneous injection of GT3. METHODS: C57BL/6 female mice were administrated GT3 (100 mg/kg body weight) with PEG-400 emulsion by subcutaneous injection. GT3 levels in different tissues were determined by HPLC with a fluorescence detector. Gene expressions were measured by real-time PCR. RESULTS: GT3 predominantly accumulated in adipose and heart tissue, and was maintained at a relatively stable level in bone tissues after a single-dose administration. Accumulated GT3 in bone tissues significantly inhibited the increase in RANKL expression and the decrease in OPG expression induced by db-cAMP. CONCLUSIONS: We investigated the tissue distribution of GT3 with PEG emulsion by subcutaneous administration, which has never been reported so far. Our results suggest that GT3 with PEG emulsion accumulated in tissues is able to carry out a long-term biological effect and has therapeutic value for treating and preventing osteoporosis.

The phthalide compound tokinolide B from Angelica sinensis exerts anti-inflammatory effects through Nur77 binding.

BACKGROUND: Nur77, an orphan member of the nuclear receptor superfamily, regulates inflammatory diseases and is a therapeutic target for treating inflammation. Phthalides in Angelica sinensis exhibit anti-inflammatory activity. PURPOSE: This study aimed to screen compounds from A. sinensis phthalide extract that could exert anti-inflammatory activity by targeting Nur77. To provide new theoretical support for better elucidation of Chinese medicine targeting mitochondria to achieve multiple clinical efficacies. METHODS: The anti-inflammatory capacity of phthalides was assessed in tumor necrosis factor-alpha (TNF-α)-stimulated HepG2 cells using western blotting. The interaction between phthalides and Nur77 was verified by molecular docking, surface plasmon resonance, and cellular thermal shift assay. Co-immunoprecipitation, western blotting, and immunostaining were performed to determine the molecular mechanisms. The in vivo anti-inflammatory activity of the phthalides was evaluated in a lipopolysaccharide (LPS)/d-galactosamine (d-GalN)-induced acute hepatitis and liver injury mouse model of acute hepatitis and liver injury. Finally, the toxicity of phthalide toxicity was assessed in zebrafish experiments. RESULTS: Among the 27 phthalide compounds isolated from A. sinensis, tokinolide B (TB) showed the best Nur77 binding capacity and, the best anti-inflammatory activity, which was induced without apoptosis. In vivo and in vitro experiments showed that TB promoted Nur77 translocation from the nucleus to the mitochondria and interacted with tumor necrosis factor receptor-associated factor 2 (TRAF2) and sequestosome 1 (p62) to induce mitophagy for anti-inflammatory functions. TB substantially inhibited LPS/d-GalN-induced acute hepatitis and liver injury in mice. TB also exhibited significantly lower toxicity than celastrol in zebrafish experiments. CONCLUSION: These findings suggested that TB inhibits inflammation by promoting Nur77 interaction with TRAF2 and p62, thereby inducing mitophagy. These findings offer promising directions for developing novel anti-inflammatory agents, enhance the understanding of phthalide compounds, and highlight the therapeutic potential of traditional Chinese herbs.

Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside I-induced EC109 cell death.

OBJECTIVE: Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu (TBM), a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma (ESCC) for a long term. tubeimoside I (TBMS1) is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells. METHODS: Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins' expression and location. RESULTS: Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMS1-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B1/cdc2 complex-related G2/M cell cycle arrest. CONCLUSIONS: Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.

A Label-Free and Antibody-Free Molecularly Imprinted Polymer-Based Impedimetric Sensor for NSCLC-Cells-Derived Exosomes Detection.

In this study, a label-free and antibody-free impedimetric biosensor based on molecularly imprinting technology for exosomes derived from non-small-cell lung cancer (NSCLC) cells was established. Involved preparation parameters were systematically investigated. In this design, with template exosomes anchored on a glassy carbon electrode (GCE) by decorated cholesterol molecules, the subsequent electro-polymerization of APBA and elution procedure afforded a selective adsorption membrane for template A549 exosomes. The adsorption of exosomes caused a rise in the impedance of the sensor, so the concentration of template exosomes can be quantified by monitoring the impedance of GCEs. Each procedure in the establishment of the sensor was monitored with a corresponding method. Methodological verification showed great sensitivity and selectivity of this method with an LOD = 2.03 × 103 and an LOQ = 4.10 × 104 particles/mL. By introducing normal cells and other cancer cells derived exosomes as interference, high selectivity was proved. Accuracy and precision were measured, with an obtained average recovery ratio of 100.76% and a resulting RSD of 1.86%. Additionally, the sensors' performance was retained at 4 °C for a week or after undergoing elution and re-adsorption cycles seven times. In summary, the sensor is competitive for clinical translational application and improving the prognosis and survival for NSCLC patients.

A resveratrol analog, phoyunbene B, induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells.

Among the seven natural resveratrol analogs separated and identified from Pholidota yunnanensis R(OLFE), we found phoyunbene B (PYB, trans-3,4'-dihydroxy-2',3',5-trimethoxystilbene) was more effective in inhibiting the growth of HepG2 hepatocellular carcinoma cells than resveratrol. The inhibitory effect of PYB in HepG2 cells was due to its induction of G2/M cell cycle arrest and apoptosis. Induction of G2/M phase cell cycle arrest by PYB was associated with its up-regulation of Cyclin B1, while its induction of apoptosis was accompanied with its down-regulation of Bcl-2 and up-regulation of Bax. Our in vitro invasion/migration assays also showed that PYB could inhibit the invasion of hepatocellular carcinoma cells.

Structural modification of ginsenoside Rh(2) by fatty acid esterification and its detoxification property in antitumor.

Ginsenoside Rh(2), one of the most important ginsenosides with anticancer properties in red ginseng, has been developed as principal antitumor ingredient for clinical use. However, the cytotoxicity test in human hepatocyte cell line QSG-7701 (IC(50) 37.3µM) indicated that Rh(2) might show strong cytotoxic side-effect on the normal liver cells. For blunting the toxicity, Rh(2) was structurally modified by reacting with octanoyl chloride to give a dioctanoyl ester of Rh(2) (D-Rh(2)) in the present study. MTT assay in QSG-7701 cell line in vitro showed that the cytotoxicity of D-Rh(2) on human hepatocyte cells (IC(50) 80.5µM) was significantly lower than that of Rh(2). While antitumor xenograft assay in mice bearing H22 liver cancer cells in vivo showed that the antitumor activity of D-Rh(2) retained to be strong as that of Rh(2). According to previous pharmacokinetic studies, the fatty acid esterification of Rh(2) might be of detoxification reaction to cells. Additionally, D-Rh(2) showed significant enhancement on increasing thymus index at the dose of 10mg/kg compared with vehicle treated control group. Thus, D-Rh(2) might indirectly affect tumor growth by stimulating lymphocytes to become cytotoxic to tumor cells. Finally, our findings suggested that D-Rh(2), the fatty acid ester of Rh(2), might attenuate the side-effect by detoxification to human normal cell and could be a more potential candidate for developing as an antitumor drug.

[Chemical constituents contained in Desmodium caudatum].

OBJECTIVE: To study chemical constituents contained in Desmodium caudatum. METHOD: The chemical compounds were separated by using such chromatographic methods as macroporous resin, Sephadex LH-20, ODS and normal phase silicagel column, and their structures were identified by spectroscopic data analysis. RESULT: Fifteen compounds were separated and identified as stigmasterol (1), beta-sitosterol (2), citrusinol (3), hibiscone A (4), yukovanol (5), kenusanone I (6), neophellamuretin (7), desmodol (8), erythrotriol (9), hibiscone D (10), kaempferol (11), 8-prenylquercetin (12), leachianone G (13), 5,7,4'-trihydroxy-dihydroflavonol (14), and 4H-1-benzopyran-4-one, 2-(3,4-dihydroxyphenyl) -2, 3-dihydro-3,5,7-trihydroxy-8-( 3-methyl-2-butenyl) -, (2R-trans)-(9CI) (15). CONCLUSION: All of the compounds were separated from D. caudatum for the first time except compound 8.

[Preliminary study on chemical constituents seperated from Cayratia japonica].

OBJECTIVE: To study effective active constituents of Cayratia japonica,a genuine herbal medicine from Fujian. METHOD: Such chromatographic methods as Macroporous, Sephadex LH-20, ODS and normal phase silica gel column chromatography were adopted to separate the chemical components of C. japonica. RESULT: Thirteen compounds were obtained, and their structures were identified by analyzing multiple spectral data as luteolin(1), apigenin(2), triethyl citrate-(3), 3-formylindole(4), esculetin(5), bis(2-ethylhexyl)-phthalate(6), calendin(7), ethyl-trans-3,4-dihydr-oxycinnamate(8), luteolin7-O-D-glucoside(9),5-hydroxy-3,4-dimethyl-5-pentyl-2(5H-furanone(10),ethyl-3,4-dihydroxybenzoate(11), eriodictyol(12) and daucosterol(13). CONCLUSION: Among them, compounds 3-8 and 10-12 were separated from the plant for the first time.

Orphan Nuclear Receptor Nur77 Mediates the Lethal Endoplasmic Reticulum Stress and Therapeutic Efficacy of Cryptomeridiol in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) commonly possesses chronical elevation of IRE1α-ASK1 signaling. Orphan nuclear receptor Nur77, a promising therapeutic target in various cancer types, is frequently silenced in HCC. In this study, we show that cryptomeridiol (Bkh126), a naturally occurring sesquiterpenoid derivative isolated from traditional Chinese medicine Magnolia officinalis, has therapeutic efficacy in HCC by aggravating the pre-activated UPR and activating the silenced Nur77. Mechanistically, Nur77 is induced to sense IRE1α-ASK1-JNK signaling and translocate to the mitochondria, which leads to the loss of mitochondrial membrane potential (Δψm). The Bkh126-induced aggravation of ER stress and mitochondrial dysfunction result in increased cytotoxic product of reactive oxygen species (ROS). The in vivo anti-HCC activity of Bkh126 is superior to that of sorafenib, currently used to treat advanced HCC. Our study shows that Bkh126 induces Nur77 to connect ER stress to mitochondria-mediated cell killing. The identification of Nur77 as a molecular target of Bhk126 provides a basis for improving the leads for the further development of anti-HCC drugs.

A novel screening model for the molecular drug for diabetes and obesity based on tyrosine phosphatase Shp2.

Tyrosine phosphatase Src-homology phosphotyrosyl phosphatase 2 (Shp2) was identified as a potential molecular target for therapeutic treatment of diabetes and obesity. However, there is still no systematic research on the enhancers for the Shp2 enzyme. The present study established a novel powerful model for the high-throughput screening of Shp2 enhancers and successfully identified a new specific Shp2 enhancer, oleanolic acid, from Chinese herbs.

Furocoumarin derivatives from radix Angelicae dahuricae and their effects on RXRα transcriptional regulation.

A novel furocoumarin derivative named oxyalloimperatorin (1), together with seventeen furocoumarins 2-18 were isolated from the radix of Angelica dahurica. The chemical structure of new metabolite was characterized by analysis of IR, NMR, and HR-ESI-MS spectroscopic data. Among the isolated compounds, 13, 16, and 18 (each at 20 µM) could significantly promote the gene transcriptional function of nuclear receptor RXRα. While 7-9, 13, 14, and the new structure 1 (each at 20 µM) showed significant reduction in RXRα gene transcriptional activities induced by 9-cis-retinoid acid. The findings indicated that these furocoumarin skeleton derivatives might hold beneficial effects on many intractable diseases, such as cancer and metabolic diseases, due to their potential activities on regulating the transcriptional activation function of RXRα.

[Chemical constituents of bear bile].

OBJECTIVE: To study the chemical constituents of bear bile. METHOD: The compounds were isolated by repeated column HP20 macroporous adsorption resin, Sephadex LH-20, ODS and silica gel as packing materials. The structures were identified on the basis of extensive spectroscopic data analysis and by comparison of their spectral data reported. RESULT: Nine compounds were identified as 4',7-dihydroxyisoflavone (1), 4',7-dihydroxy-6-methoxyisoflavone (2), 4',6,7-trihydroxyisoflavone (3), 4'-methoxy-7-hydroxyisoflavone (4), tauroursodeoxycholic acid (5), taurochenodeoxycholic acid (6), ursodeoxycholic acid (7), chenodeoxycholic acid (8), cholesterol (9). CONCLUSION: Compounds 1-4 were separated from bear bile for the first time.

[Steroids isolated from fruit of Hovenia acerba].

OBJECTIVE: To study on the chemical constituents of Hovenia acerba. METHODS: Compounds were isolated with stratography and solvent, and identified by spectroscopy. RESULT: beta-sitoseterol, 3 beta-hydroxy-3-deoxymoronic acid and beta-sitoseteryl-3-O-beta-D-glucopyranoside were obtained from fruit of Hovenia acerba. CONCLUSION: The three compounds were steroids isolated from Hovenia acerba Thunb. for the frist time.

[Comparative analysis of ginsenosides from red ginseng].

OBJECTIVE: To determine ginsenoside Rg1, Re, Rb1, Rc, Rb2, Rd from main root and root hair of red ginseng of different specifications. METHOD: Ultrasonical extraction and reversed phase high performance liquid chromatography were applied. RESULT: The total contents of six ginsenosides from main root 15 roots (percent 500 g), 20 roots, 30 roots and root hair are 1.21%, 1.46%, 1.54% and 8.16%, respectively. CONCLUSION: The results showed that the bigger the volume of ginseng root, the less the content of ginsenoside.

Online polar two phase countercurrent chromatography×high performance liquid chromatography for preparative isolation of polar polyphenols from tea extract in a single step.

Herein, we report an on-line two-dimensional system constructed by counter-current chromatography (CCC) coupling with preparative high-performance liquid chromatography (prep-HPLC) for the separation and purification of polar natural products. The CCC was used as the first dimensional isolation column, where an environmental friendly polar two-phase solvent system of isopropanol and 16% sodium chloride aqueous solution (1:1.2, v/v) was introduced for low toxicity and favorable resolution. In addition, by applying the stop-and-go flow technique, effluents pre-fractionated by CCC was further purified by a preparative column packed with octadecyl silane (ODS) as the second dimension. The interface between the two dimensions was comprised of a 6-port switching valve and an electronically controlled 2-position 10-port switching valve connected with two equivalent holding columns. To be highlighted here, this rationally designed interface for the purpose of smooth desalination, absorption and desorption, successfully solved the solvent compatibility problem between the two dimensional separation systems. The present integrated system was successfully applied in a one-step preparative separation and identification of 10 pure compounds from the water extracts of Tieguanyin tea (Chinese oolong tea). In short, all the results demonstrated that the on-line 2D CCC×LC method is an efficient and green approach for harvesting polar targets in a single step, which showed great promise in drug discovery.

Pyrroloquinoline quinone prevents MK-801-induced stereotypical behavior and cognitive deficits in mice.

Pyrroloquinoline quinone (PQQ), an essential nutrient, antioxidant, redox modulator, and nerve growth factor, prevents cognitive deficits associated with oxidative stress-induced neurodegeneration. Previous molecular imaging studies also demonstrate that PQQ binds to N-methyl D-aspartate (NMDA) receptors. In this study, we investigated the effects of PQQ on stereotypical behaviors and cognitive deficits induced by MK-801, a non-competitive NMDA antagonist used to model schizophrenia. Mice were given repeated injections of MK-801 (0.5mg/kg/d) and PQQ (0.2, 2.0, or 20 µg/kg/d) for 60 days. Behavior was evaluated using a variety of motor, social, and cognitive tests. We found that PQQ administration significantly attenuated MK-801-induced increases in stereotypical behavior and ataxia, suggesting a protective role of PQQ against MK-801-induced neuronal dysfunction and psychiatric disorders. Future studies are necessary to elucidate the underlying mechanisms of PQQ.

Comparative study of somatostatin-human serum albumin fusion proteins and natural somatostatin on receptor binding, internalization and activation.

Albumin fusion technology, the combination of small molecular proteins or peptides with human serum albumin (HSA), is an effective method for improving the medicinal values of natural small molecular proteins or peptides. However, comparative studies between HSA-fusion proteins or peptides and the parent small molecules in biological and molecular mechanisms are less reported. In this study, we examined the binding property of two novel somatostatin-HSA fusion proteins, (SST14)2-HSA and (SST28)2-HSA, to human SSTRs in stably expressing SSTR1-5 HEK 293 cells; observed the regulation of receptor internalization and internalized receptor recycling; and detected the receptors activation of HSA fusion proteins in stably expressing SSTR2- and SSTR3-EGFP cells. We showed that both somatostatin-HSA fusion proteins had high affinity to all five SSTRs, stimulated the ERK1/2 phosphorylation and persistently inhibited the accumulation of forskolin-stimulated cAMP in SSTR2- and SSTR3-expressing cells; but were less potent than the synthetic somatostatin-14 (SST-14). Our experiments also showed that somatostatin-HSA fusion proteins did not induce the receptors internalization; rather, they accelerated the recycling of the internalized receptors induced by SST-14 to the plasma membrane. Our results indicated that somatostatin-HSA fusion proteins, different from SST-14, exhibit some particular properties in binding, regulating, and activating somatostatin receptors.

Raddeanalin, a new flavonoid glycoside from the leaves of Salix raddeana Laksh.

A new flavonoid glycoside, diosmetin 7-O-beta-D-xylopyranosyl(1-6)-beta-D-glucopyranoside, named raddeanalin (1), was isolated from the ethanolic extract of the leaves of Salix raddeana Laksh. together with three known compounds, kaempferol 3-O-glucoside (2), quercetin 3-O-glucoside (3) and quercetin 3-O-rutinoside (4). The structure of new compound was established by the spectral data and chemical properties.