Raltitrexed increases radiation sensitivity of esophageal squamous carcinoma cells.

BACKGROUND: Radiation therapy remains an important therapeutic modality, especially for those patients who are not candidates for radical resection. Many strategies have been developed to increase the radiosensitivity of esophageal cancer, with some success. METHODS: This study was conducted to determine whether raltitrexed can enhance radiosensitivity of esophageal squamous cell carcinoma (ESCC). ESCC cell lines 24 h were incubated with raltitrexed or DMSO with or without subsequent irradiation. Cell Counting Kit assay-8 assay and clonogenic survival assay were used to measure the cell proliferation and radiosensitization, respectively. Flow cytometry was utilized to examine cell apoptosis and cell cycle distribution in different groups. Immunofluorescence analysis was performed to detect deoxyribonucleic acid (DNA) double-strand breaks. In addition, the expression levels of proteins that are involved in radiation induced signal transduction including Bax, Cyclin B1, Cdc2/pCdc2, and Cdc25C/pCdc25C were examined by western blot analysis. RESULTS: The results indicated that raltitrexed enhanced radiosensitivity of ESCC cells with increased DNA double-strand breaks, the G2/M arrest, and the apoptosis of ESCC cells induced by radiation. The sensitization enhancement ratio of 1.23-2.10 was detected for ESCC cells with raltitrexed treatment in TE-13 cell line. In vitro, raltitrexed also increased the therapeutic effect of radiation in nude mice. CONCLUSION: Raltitrexed increases the radiosensitivity of ESCC. This antimetabolite drug is promising for future clinical trials with concurrent radiation in esophageal cancer.

Bisdemethoxycurcumin sensitizes cisplatin-resistant lung cancer cells to chemotherapy by inhibition of CA916798 and PI3K/AKT signaling.

Curcumin, a dietary supplement or herbal medicine from Curcuma longa, has shown antitumor activity in different cancer cell lines and clinical trials. CA916798, a novel protein, is overexpressed in multidrug-resistant tumor cells. This study aimed to assess the effects of curcumin on regulating chemosensitivity in cisplatin-resistant non-small cell lung cancer (NSCLC) cells in vitro and to explore the underlying molecular mechanisms. Human cisplatin-sensitive A549 and cisplatin-resistant A549/CDDP lung adenocarcinoma cells were treated with curcumin to assess cell viability and gene modulations using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. CA916798 shRNA and point mutations were used to assess the CA916798 functions and phosphorylation sites. Bisdemethoxycurcumin sensitized cisplatin-resistant lung cancer cells to various chemotherapeutic agents, including cisplatin. Bisdemethoxycurcumin reduced the levels of CA916798 mRNA and protein in A549 and A549/CDDP cells, while it also suppressed phosphatidylinositol-3-kinase (PI3K)/AKT signaling. CA916798, as a downstream gene, interacted with AKT after bisdemethoxycurcumin treatment in A549 and A549/CDDP cells. Moreover, A549/CDDP cells expressing the point-mutated CA916798-S20D protein were more resistant to cisplatin and bisdemethoxycurcumin, whereas tumor cells expressing CA916798-S20A, CA916798-S31A, CA916798-S60A, CA916798-S93A, or CA916798-T97A (different sites of amino acid phosphorylation) showed similar sensitivity or resistance to cisplatin and bisdemethoxycurcumin, compared with the control cells. Bisdemethoxycurcumin is able to sensitize cisplatin-resistant NSCLC cells to chemotherapeutic agents by inhibition of CA916798 and PI3K/AKT activities. Moreover, phosphorylation of CA916798 at the S20 residue plays a critical role in mediating bisdemethoxycurcumin antitumor activity.

Autophagy Accompanied with Bisdemethoxycurcumin-induced Apoptosis in Non-small Cell Lung Cancer Cells.

OBJECTIVE: To investigate the effects of bisdemethoxycurcumin (BDMC) on non-small cell lung cancer (NSCLC) cell line, A549, and the highly metastatic lung cancer 95D cells. METHODS: CCK-8 assay was used to assess the effect of BDMC on cytotoxicity. Flow cytometry was used to evaluate apoptosis. Western blot analysis, electron microscopy, and quantification of GFP-LC3 punctuates were used to test the effect of BDMC on autophagy and apoptosis of lung cancer cells. RESULTS: BDMC inhibited the viability of NSCLC cells, but had no cytotoxic effects on lung small airway epithelial cells (SAECs). The apoptotic cell death induced by BDMC was accompanied with the induction of autophagy in NSCLC cells. Blockage of autophagy by the autophagy inhibitor 3-methyladenine (3-MA) repressed the growth inhibitory effects and induction of apoptosis by BDMC. In addition, BDMC treatment significantly decreased smoothened (SMO) and the transcription factor glioma-associated oncogene 1 (Gli1) expression. Furthermore, depletion of Gli1 by siRNA and cyclopamine (a specific SMO inhibitor) induced autophagy. CONCLUSION: Aberrant activation of Hedgehog (Hh) signaling has been implicated in several human cancers, including lung cancers. The present findings provide direct evidence that BDMC-induced autophagy plays a pro-death role in NSCLC, in part, by inhibiting Hedgehog signaling.

Lewis base-promoted rearrangement of allylic cyanohydrins: construction of functionalized nitriles bearing 1,3-diketone moieties.

A novel Lewis base-promoted rearrangement of allylic cyanohydrins has been developed, in which the cyano group was rearranged, directly coupled with the generation of new functional groups. This protocol provides a unique and facile way to prepare highly functionalized nitriles bearing 1,3-diketone moieties under mild reaction conditions. Furthermore, the synthetic transformations of the functionalized products have also been demonstrated.

[Studies on the constituents from the herba of Erigeron acer].

OBJECTIVE: To investigate the chemical constituents of the aerial part of Erigeron acer. METHODS: The chemical constituents were isolated by various columns and thin layer chromatographic methods. The structures were identified by spectral data. RESULTS: Five compounds were isolated and identified as alpha-amyrin (1), beta-amyrin (2), caffeic acid (3), quercetin (4), 4'-hydroxywogonin-7-O-beta-D-glucuronic acid glycoside (5). CONCLUSION: The five compounds are obtained from this plant for the first time. The signals of 13C-NMR of the compound(5) are reassigned by means of DEPT, HMQC, HMBC, the signals of 1H-NMR of 2" - 5"-H of the glucuronic acid moiety are assigned by means of HMBC, HMQC, 1H-1H COSY for the first time.

[Chemical Compositions and Source Apportionment of Road Dust in Yuncheng].

Samples of particulate sources in Yuncheng including road dust, salt lake dust, coal dust, soil dust, construction,cement dust and vehicle exhaust dust were collected. Elements, ions and carbon species in particulate sources samples were analyzed. Enrichment factors and potential ecological risk assessment were used to analyze the characteristics of road dust, and chemical mass balance model was applied to identify the source of road dust. The results showed that, compared with other cities, the proportions of Na(12.1970%) and SO42-(8.5971%) were relatively high while that of Si(9.1123%) was low in road dust in Yuncheng, and enrichment factors showed that the sources of Pb, Cu, Cr, V, As, Ni, Na and Zn in road dust were obviously influenced by human activities; the potential ecological risk of heavy metals in road dust was high, which was affected by anthropogenic sources such as industrial production, the combustion of fossil fuels and vehicle exhaust; the profiles of coal dust, vehicle exhaust dust, construction and cement dust were similar to those of other cities, the Na and SO42- concentrations in soil dust were relatively high, and the proportions of Na and SO42- in salt lake dust were 30.3% and 22.7% respectively; salt lake dust was the largest contributor (53%) to road dust, followed by the soil dust (21%), vehicle exhaust dust (8%), construction and cement dust (7%), and coal dust (5%).

Copper-Promoted Cyclization of α-Amino Nitrile-Tethered Enynes: Controllable Synthesis of 3-Azabicyclo[4.1.0]hepta-2,4-dienes and 4,5-Dihydro-3H-azepines.

The first example of Cu-promoted cyclization of α-amino nitrile-tethered enynes incorporating an electron-deficient alkene component is described. A wide range of functionalized 3-azabicyclo[4.1.0]hepta-2,4-dienes and 4,5-dihydro-3H-azepines were prepared efficiently in a controllable manner. Moreover, the diverse cascade process enables efficient incorporation of tertiary amine moieties under mild reaction conditions. A possible reaction pathway is proposed on the basis of a series of control experiments.

Room-temperature in situ nuclear spin hyperpolarization from optically pumped nitrogen vacancy centres in diamond.

Low detection sensitivity stemming from the weak polarization of nuclear spins is a primary limitation of magnetic resonance spectroscopy and imaging. Methods have been developed to enhance nuclear spin polarization but they typically require high magnetic fields, cryogenic temperatures or sample transfer between magnets. Here we report bulk, room-temperature hyperpolarization of (13)C nuclear spins observed via high-field magnetic resonance. The technique harnesses the high optically induced spin polarization of diamond nitrogen vacancy centres at room temperature in combination with dynamic nuclear polarization. We observe bulk nuclear spin polarization of 6%, an enhancement of ∼170,000 over thermal equilibrium. The signal of the hyperpolarized spins was detected in situ with a standard nuclear magnetic resonance probe without the need for sample shuttling or precise crystal orientation. Hyperpolarization via optical pumping/dynamic nuclear polarization should function at arbitrary magnetic fields enabling orders of magnitude sensitivity enhancement for nuclear magnetic resonance of solids and liquids under ambient conditions.

Role of Wnt Inhibitory Factor-1 in Inhibition of Bisdemethoxycurcumin Mediated Epithelial-to-Mesenchymal Transition in Highly Metastatic Lung Cancer 95D Cells.

BACKGROUND: Bisdemethoxycurcumin (BDMC) is an active component of curcumin and a chemotherapeutic agent, which has been suggested to inhibit tumor growth, invasion and metastasis in multiple cancers. But its contribution and mechanism of action in invasion and metastasis of non-small cell lung cancer (NSCLC) are not very clear. Therefore, we tried to study the effects of BDMC on regulation of epithelial-to-mesenchymal transition (EMT), which is closely linked to tumor cell invasion and metastasis. METHODS: In this study, we first induced transforming growth factor-ß1 (TGF-ß1) mediated EMT in highly metastatic lung cancer 95D cells. Thereafter, we studied the effects of BDMC on invasion and migration of 95D cells. In addition, EMT markers expressions were also analyzed by western blot and immunofluorescence assays. The contribution of Wnt inhibitory factor-1 (WIF-1) in regulating BDMC effects on TGF-ß1 induced EMT were further analyzed by its overexpression and small interfering RNA knockdown studies. RESULTS: It was observed that BDMC inhibited the TGF-ß1 induced EMT in 95D cells. Furthermore, it also inhibited the Wnt signaling pathway by upregulating WIF-1 protein expression. In addition, WIF-1 manipulation studies further revealed that WIF-1 is a central molecule mediating BDMC response towards TGF-ß1 induced EMT by regulating cell invasion and migration. CONCLUSIONS: Our study concluded that BDMC effects on TGF-ß1 induced EMT in NSCLC are mediated through WIF-1 and elucidated a novel mechanism of EMT regulation by BDMC.

Optically detected cross-relaxation spectroscopy of electron spins in diamond.

The application of magnetic resonance spectroscopy at progressively smaller length scales may eventually permit 'chemical imaging' of spins at the surfaces of materials and biological complexes. In particular, the negatively charged nitrogen-vacancy (NV(-)) centre in diamond has been exploited as an optical transducer for nanoscale nuclear magnetic resonance. However, the spectra of detected spins are generally broadened by their interaction with proximate paramagnetic NV(-) centres through coherent and incoherent mechanisms. Here we demonstrate a detection technique that can resolve the spectra of electron spins coupled to NV(-) centres, in this case, substitutional nitrogen and neutral nitrogen-vacancy centres in diamond, through optically detected cross-relaxation. The hyperfine spectra of these spins are a unique chemical identifier, suggesting the possibility, in combination with recent results in diamonds harbouring shallow NV(-) implants, that the spectra of spins external to the diamond can be similarly detected.

Akt1 enhances CA916798 expression through mTOR pathway.

Multi-drug resistance leads to the failure of chemotherapy for cancers. Our previous study showed that overexpression of CA916798 led to multi-drug resistance. However, the underlying mechanisms remain unknown. In the current study, we observed that the levels of phosphorylated AKT, phosphorylated mTOR and CA916798 all increased in the drug resistant human adenocarcinoma samples and paralleled with the change of drug resistance. The results of immunofluorescence and Co-IP indicated that the positive correlation of CA916798 expression with AKT1 activation might be associated with drug resistance of lung adenocarcinoma. Furthermore, AKT1 stimulated CA916798 expression through mTOR pathway in both A549 and A549/CDDP cell lines, which was also observed in the xenografted tumor in nude mice. The results showed that CA916798 located in the downstream of PI3K/AKT/mTOR pathway. Inhibition of PI3K by LY294002 could efficiently reduce CA916798 expression and tumor size in vivo as well. Additionally, LY294002 combined with rapamycin inhibited CA916798 expression and tumor size stronger than LY294002 alone. Our findings may also provide a new explanation for synergistic anti-tumor effects of PI3K and mTORC1 inhibitors.

Critical role of water in the binding of volatile anesthetics to proteins.

Numerous small molecules exhibit drug-like properties by low-affinity binding to proteins. Such binding is known to be influenced by water, the detailed picture of which, however, remains unclear. One particular example is the controversial role of water in the binding of general anesthetics to proteins as an essential step in general anesthesia. Here we demonstrate that a critical amount of hydration water is a prerequisite for anesthetic-protein binding. Using nuclear magnetic resonance, the concurrent adsorption of hydration water and bound anesthetics on model proteins are simultaneously measured. Halothane binding on proteins can only take place after protein hydration reaches a threshold hydration level of ∼0.31 g of water/g of proteins at the relative water vapor pressure of ∼0.95. Similar dependence on hydration is also observed for several other anesthetics. The ratio of anesthetic partial pressures at which two different anesthetics reach the same fractional load is correlated with the anesthetic potency. The binding of nonimmobilizers, which are structurally similar to known anesthetics but unable to produce anesthesia, does not occur even after the proteins are fully hydrated. Our results provide the first unambiguous experimental evidence that water is absolutely required to enable anesthetic-protein interactions, shedding new light on the general mechanism of molecular recognition and binding.

Sensitive magnetic control of ensemble nuclear spin hyperpolarization in diamond.

Dynamic nuclear polarization, which transfers the spin polarization of electrons to nuclei, is routinely applied to enhance the sensitivity of nuclear magnetic resonance. This method is particularly useful when spin hyperpolarization can be produced and controlled optically or electrically. Here we show complete polarization of nuclei located near optically polarized nitrogen-vacancy centres in diamond. Close to the ground-state level anti-crossing condition of the nitrogen-vacancy electron spins, (13)C nuclei in the first shell are polarized in a pattern that depends sensitively upon the magnetic field. Based on the anisotropy of the hyperfine coupling and of the optical polarization mechanism, we predict and observe a reversal of the nuclear spin polarization with only a few millitesla change in the magnetic field. This method of magnetic control of high nuclear polarization at room temperature can be applied in sensitivity enhanced nuclear magnetic resonance of bulk nuclei, nuclear-based spintronics, and quantum computation in diamond.

Temperature dependence of lysozyme hydration and the role of elastic energy.

Water plays a critical role in protein dynamics and functions. However, the most basic property of hydration--the water sorption isotherm--remains inadequately understood. Surface adsorption is the commonly adopted picture of hydration. Since it does not account for changes in the conformational entropy of proteins, it is difficult to explain why protein dynamics and activity change upon hydration. The solution picture of hydration provides an alternative approach to describe the thermodynamics of hydration. Here, the flexibility of proteins could influence the hydration level through the change of elastic energy upon hydration. Using nuclear magnetic resonance to measure the isotherms of lysozyme in situ between 18 and 2 °C, the present work provides evidence that the part of water uptake associated with the onset of protein function is significantly reduced below 8 °C. Quantitative analysis shows that such reduction is directly related to the reduction of protein flexibility and enhanced cost in elastic energy upon hydration at lower temperature. The elastic property derived from the water isotherm agrees with direct mechanical measurements, providing independent support for the solution model. This result also implies that water adsorption at charged and polar groups occurring at low vapor pressure, which is known for softening the protein, is crucial for the later stage of water uptake, leading to the activation of protein dynamics. The present work sheds light on the mutual influence of protein flexibility and hydration, providing the basis for understanding the role of hydration on protein dynamics.

CA916798 regulates multidrug resistance of lung cancer cells.

OBJECTIVES: Multidrug resistance (MDR) significantly reduces the efficacy of chemotherapy for lung cancer. In this study, we characterized the significance of CA916798, a gene up-regulated in cis-dichlorodiamine platinum (CDDP)-resistant lung adenocarcinoma cells, in mediating MDR in lung cancer cells. METHODS: CA916798 was stably transfected into H446 cells with low endogenous expression of CA916798, and knocked down in A549/CDDP cells with high endogenous level of CA916798. Expression was confirmed by real-time PCR, Western immunoblotting and immunocytochemistry. Subsequent effects were examined on cellular growth, apoptosis and cell cycle progression. RESULTS: Ectopic expression of CA916798 in H446 cells confered enhanced resistance to multiple chemotherapeutic agents, while its reduction rendered A549/CDDP cells less resistant to chemotherapeutic agents tested. Further analysis revealed that CA916798 regulates CDDP-induced cell growth, apoptosis and cell cycle progression. CONCLUSION: CA916798 may be a novel MDR-related target for lung cancer therapy.

Hypomethylation effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin on WIF-1 promoter in non-small cell lung cancer cell lines.

The tumor suppressor gene Wnt inhibitory factor-1 (WIF-1) has been found to be promoter hypermethylated and silenced in lung cancer cell lines and tissues. Curcuminoids are major active components of the spice turmeric, and have recently been reported to be potential hypomethylation agents. In the present study, the hypomethylation effects of three major curcuminoids, curcumin, demethoxycurcumin and bisdemethoxycurcumin, were compared in vitro using ELISA, and their demethylation potential was confirmed by methylation-specific PCR. It was found that bisdemethoxycurcumin possesses the strongest demethylation function in vitro compared to the other two curcuminoids, exerting its effect at a minimal demethylation concentration of 0.5-1 µM. The WIF-1 promoter region was demethylated after treatment with 20 µM demethoxycurcumin and bisdemethoxycurcumin, but failed to respond to 20 µM curcumin. In the A549 cell line, RT-PCR and Western blotting were used to confirm that WIF-1 expression was restored after curcuminoid-induced promoter hypermethylation. Since the results regarding the demethylation potential of the three major curcuminoids to restore WIF-1 expression indicated that bisdemethoxycurcumin has the strongest hypomethylation effect, this curcuminoid may have therapeutic use in the restoration of WIF-1 expression in NSCLC.

Temperature-induced hydrophobic-hydrophilic transition observed by water adsorption.

The properties of nanoconfined and interfacial water in the proximity of hydrophobic surfaces play a pivotal role in a variety of important phenomena such as protein folding. Water inside single-walled carbon nanotubes (SWNTs) can provide an ideal system for investigating such nanoconfined interfacial water on hydrophobic surfaces, provided that the nanotubes can be opened without introducing excess defects. Here, we report a hydrophobic-hydrophilic transition upon cooling from 22 degrees C to 8 degrees C via the observation of water adsorption isotherms in SWNTs measured by nuclear magnetic resonance. A considerable slowdown in molecular reorientation of such adsorbed water was also detected. The observed transition demonstrates that the structure of interfacial water could depend sensitively on temperature, which could lead to intriguing temperature dependences involving interfacial water on hydrophobic surfaces.

Identification of endohedral water in single-walled carbon nanotubes by (1)H NMR.

Water confinement within single-walled carbon nanotubes (SWCNTs) has been a topic of current interest, due in part to their potential nanofiltration applications. Experiments have recently validated molecular dynamics predictions of flow enhancement within these channels, although few studies have probed the detailed structure and dynamics of water in these systems. Proton nuclear magnetic resonance ( (1)H NMR) is a technique capable of providing some of these details, although care must be exercised in separating the confined water of interest from exterior water. By using controlled experiments with both sealed and opened SWCNTs and by providing a quantitative measure of water content through desorption experiments, a signature for confined water in SWCNTs has been positively identified. This endohedral or interior water is characterized by a relatively broad feature located at 0.0 ppm, shifted upfield relative to bulk water. With the identification of a signature for water inside SWCNTs, further studies aimed at probing water dynamics will be enabled.