Ouabain impairs cancer metabolism and activates AMPK-Src signaling pathway in human cancer cell lines.

In addition to the well-known cardiotonic effects, cardiac glycosides (CGs) produce potent anticancer effects with various molecular mechanisms. We previously show that ouabain induces autophagic cell death in human lung cancer cells by regulating AMPK-mediated mTOR and Src-mediated ERK1/2 signaling pathways. However, whether and how AMPK and Src signaling interacts in ouabain-treated cancer cells remains unclear. Given the pivotal role of AMPK in metabolism, whether ouabain affects cancer cell metabolism remains elusive. In this study we showed that treatment with ouabain (25 nM) caused simultaneous activation of AMPK and Src signaling pathways in human lung cancer A549 cells and human breast cancer MCF7 cells. Cotreatment with AMPK inhibitor compound C or siRNA greatly abrogates ouabain-induced Src activation, whereas cotreatment with Src inhibitor PP2 has little effect on ouabain-induced AMPK activity, suggesting that AMPK served as an upstream regulator of the Src signaling pathway. On the other hand, ouabain treatment greatly depletes ATP production in A549 and MCF7 cells, and supplement of ATP (100 µM) blocked ouabain-induced AMPK activation. We further demonstrated that ouabain greatly inhibited the mitochondrial oxidative phosphorylation (OXPHOS) in the cancer cells, and exerted differential metabolic effects on glycolysis depending on cancer cell type. Taken together, this study reveals that the altered cancer cell metabolism caused by ouabain may contribute to AMPK activation, as well as its cytotoxicity towards cancer cells.

Is the combinational administration of doxorubicin and glutathione a reasonable proposal?

The combinational administration of antioxidants and chemotherapeutic agents during conventional cancer treatment is among one of the most controversial areas in oncology. Although the data on the combinational usage of doxorubicin (DOX) and glutathione (GSH) agents have been explored for over 20 years, the duration, administration route, and authentic rationality have not yet been fully understood yet. In the current study, we systematically investigated the pharmacokinetics (PK) and pharmacodynamics (PD) with both in vivo and in vitro models to elucidate the influence of GSH on the toxicity and efficacy of DOX. We first studied the cardioprotective and hepatoprotective effects of GSH in Balb/c mice, H9c2, and HL7702 cells. We showed that coadministration of exogenous GSH (5, 50, and 500 mg/kg per day, intragastric) significantly attenuated DOX-induced cardiotoxicity and hepatotoxicity by increasing intracellular GSH levels, whereas the elevated GSH concentrations did not affect the exposure of DOX in mouse heart and liver. From PK and PD perspectives, then the influences of GSH on the chemotherapeutic efficacy of DOX were investigated in xenografted nude mice and cancer cell models, including MCF-7, HepG2, and Caco-2 cells, which revealed that administration of exogenous GSH dose-dependently attenuated the anticancer efficacy of DOX in vivo and in vitro, although the elevated GSH levels neither influenced the concentration of DOX in tumors in vivo, nor the uptake of DOX in MCF-7 tumor cells in vitro. Based on the results we suggest that the combined administration of GSH and DOX should be contraindicated during chemotherapy unless DOX has caused serious hepatotoxicity and cardiotoxicity.

Metabolic integration of azide functionalized glycan on Escherichia coli cell surface for specific covalent immobilization onto magnetic nanoparticles with click chemistry.

A method for specific immobilization of whole-cell with covalent bonds was developed through a click reaction between alkyne and azide groups. In this approach, magnetic nanoparticle Fe3O4@SiO2-NH2-alkyne was synthesized with Fe3O4 core preparation, SiO2 coating, and alkyne functionalization on the surface. The azides were successfully integrated onto the cell surface of the recombinant E. coli harboring glycerol dehydrogenase, which was employed as the model cell. The highest immobilization yield of 83% and activity recovery of 94% were obtained under the conditions of 0.67 mg mg-1 cell-support ratio, pH 6.0, temperature 45 °C, and 20 mM Cu2+ concentration. The immobilized cell showed good reusability, which remained over 50% of initial activity after 10 cycles of utilization. Its activity was 9.7-fold higher than that of the free cell at the condition of pH 8.0 and each optimal temperature. Furthermore, the immobilized cell showed significantly higher activity, operational stability, and reusability.

[An association between glucose metabolism status and brachial-ankle pulse wave velocity in Chinese patients with coronary artery disease].

OBJECTIVE: To investigate the association between glucose metabolism status and brachial-ankle pulse wave velocity (baPWV) in Chinese patients with coronary artery disease (CAD). METHODS: baPWV values were measured in 198 CAD patients. Plasma glucose, serum lipids, hsCRP and other baseline data were obtained in all the patients. According to the glucose level, the participants were divided into 3 groups: normal glucose tolerance (NGT) group, impaired glucose regulation (IGR) group and diabetes mellitus (DM) group. RESULTS: baPWV values in the DM group (1807 +/- 381) cm/s were significantly higher than those in the NGT group [(1615 +/- 248) cm/s, P = 0.000] and IGR group [(1674 +/- 277) cm/s, P = 0.035]. Multiple stepwise linear regression analyses indicated that higher baPWV values were associated with aging and hsCRP levels in 198 patients. In DM group, higher baPWV values were independently associated with aging and HbA1c levels. CONCLUSIONS: In patients with coronary artery disease, baPWV values increase with different glucose metabolism status from NGT to IGR then DM. baPWV values in the patients with DM were significantly higher than those in the other two groups. Glycemic control may ameliorate arterial stiffness.

Bupivacaine in the emergency department is underused: scope for improved patient care.

AIMS: To determine patterns of local anaesthetic use, knowledge and perceived use of local anaesthetic by emergency department doctors, and barriers to bupivacaine use. METHODS: This was a multifaceted, observational study undertaken at two large metropolitan emergency departments. It comprised a retrospective chart review of patients who had been given local anaesthetic in the emergency department, an examination of ordering records of local anaesthetics in the emergency department, and a cross-sectional survey of emergency department doctors. RESULTS: The charts of 95 patients were reviewed. Most (93.7%) injuries were lacerations and the most common site was the hand (41.4%). 88 (92.6%), 4 (4.2%) and 3 (3.2%) patients were given lignocaine, prilocaine (Bier's blocks) and bupivacaine (digital blocks), respectively. Four (4.2%) cases were identified for which bupivacaine was likely to have been a better alternative than the lignocaine used. These were finger/hand injuries likely to be associated with considerable prolonged pain. The emergency department pharmacy records indicated that 30 times more lignocaine than bupivacaine was ordered in 2004-5. 30 (88.2%) of 34 doctors completed the survey. Knowledge of local anaesthetic pharmacology was variable: 33% and 66% did not know that bupivacaine was more cardiotoxic and that lignocaine was more painful, respectively. The main barriers to bupivacaine use were "habit" of using lignocaine (46.7%), cardiac toxicity (40%) and slower onset (30%). CONCLUSION: Bupivacaine seems to be underused in some appropriate circumstances. Accordingly, there is scope for improvement in patient care through critical evaluation of local anaesthetic practice. This is particularly necessary because barriers to bupivacaine use are often non-clinical (habit, availability, familiarity) rather than clinical (toxicity, onset time).

Using concanavalinA as a spacer for immobilization of E. coli onto magnetic nanoparticles.

ConcanavalinA (conA) is a protein extracted from the concanavalin, which has specific recognition through mannose components on bacterial cell surfaces. A magnetic nanocarrier with the structure of a dopamine functionalized magnetic nanoparticles was grafted with conA, and was used for immobilization of recombinant Escherichia coli harboring glycerol dehydrogenase with the specific recognition between glycoconjugates and glycoprotein. The effect of various factors on the immobilization including temperature, pH, cell concentration and immobilization time were investigated. The highest immobilization yield of 91% was obtained under the conditions: enzyme/support 1.28mg/mg, pH 8.0, immobilization time 2h and temperature 4°C. The obtained immobilized cell was characterized and exhibited higher thermal stability compared with the free cell. After ten cycles, the immobilized cell remained 62% initial activity. These results indicate that the cell immobilized onto conA-grafted nanoparticles by specific recognition of glycoconjugates and glycoprotein is a potential method for preparation of stable cell, and the immobilized cell showed perspective applications in the biocatalysis and biosensors.

Design, synthesis and biological evaluation of novel benzimidazole-2-substituted phenyl or pyridine propyl ketene derivatives as antitumour agents.

A series of novel benzimidazole-2-subsituted phenyl or pyridine propyl ketene derivatives were designed and synthesized. The biological activities of these derivatives were then evaluated as potential antitumour agents. These compounds were assayed for growth-inhibitory activity against HCT116, MCF-7 and HepG2 cell lines in vitro. The IC50 values of compounds A1 and A7 against the cancer cells were 0.06-3.64 µM and 0.04-9.80 µM, respectively. Their antiproliferative activities were significantly better than that of 5-Fluorouracil (IC50: 56.96-174.50 µM) and were close to that of Paclitaxel (IC50: 0.026-1.53 µM). The activity of these derivatives was over 100 times more effective than other reported structures of chalcone analogues (licochalcone A). A preliminary mechanistic study suggested that these compounds inhibit p53-MDM2 binding. Compounds A1, A7 and A9 effectively inhibited tumour growth in BALB/c mice with colon carcinoma HCT116 cells. The group administered 200 mg/kg of compound A7 showed a 74.6% tumour growth inhibition with no signs of toxicity at high doses that was similar to the inhibition achieved with the 12.5 mg/kg irinotecan positive control (70.2%). Therefore, this class of benzimidazole-2-subsituted phenyl or pyridine propyl ketene derivatives represents a promising lead structure for the development of possible p53-MDM2 inhibitors as new antitumour agents.

Gold conjugate-based liposomes with hybrid cluster bomb structure for liver cancer therapy.

Hybrid drug delivery system containing both organic and inorganic nanocarriers is expected to achieve its complementary advantages for the aim of improving the performance of antineoplastic drugs in tumor therapy. Here we report the use of liposomes and gold nanoparticles to construct a liposome with a hybrid Cluster Bomb structure and discuss its unique multi-order drug release property for liver tumor treatment. A very simple method is used for the hybrid liposome preparation and involves mixing two solutions containing liposomes loaded with either non-covalent or covalent Paclitaxel (PTX, namely free PTX or PTX-conjugated GNPs, respectively) by different ratio of volume (25:75, 50:50, 25:75, v/v). Various mixed liposomes were tested to determine the optimal conditions for maximum drug delivery. The optimized liposome was then tested using xenograft Heps tumor-bearing mice and showed the best efficacy for chemotherapeutic inhibition of tumor at PTX liposome: PTX-conjugated GNP liposome of 25:75 ratio (v/v). This system allows for simple and easy preparation while providing a more accurate site- and time-release mode for tumor treatment using antitumor drugs.

Synthesis and biological evaluation of geldanamycin analogs against human cancer cells.

PURPOSE: To find novel potential and less toxic benquinone anamycin heat shock protein 90 (Hsp90) inhibitors as anticancer agents, a limited series of 17-substituted or 17,19-disubstituted 17-demethoxygeldanamycin analogs were synthesized and tested for anti-proliferation activity against human cancer cells. Liver toxicity was also tested in vivo. METHODS: Five 17-alkylamino-17-demethoxygeldanamycins and two 17-alkylamino-19-methylthio-17-demethoxygeldanamycins were synthesized from geldanamycin (GA) and 19-methylthiogeldanamycin (19-S(methyl)-GA), respectively. Anti-proliferation activities of the GA analogs were determined in MCF7, HeLa, HCT116 and HepG2 cells using the MTT method. Western blot and cell cycle analyses were performed for mechanistic study. The growth inhibition effect of potential geldanamycins was also investigated in normal Buffalo rat liver (BRL) cells. In vivo liver toxicity was tested in Institute of Cancer Research (ICR) mice by tail vein injection of the tested compounds. RESULTS: Most of the 17-alkylaminogeldanamycins exhibited obvious growth inhibition effects on multiple human cancer cell lines. The anti-proliferation activity of 19-methylthio-substituted geldanamycins was significantly lower compared with no 19-substitution geldanamycins in all tested cancer cells. The compound 1b (17-[2-(piperidinyl-1'-yl)-ethylamino]-17-demethoxygeldanamycin) exhibited the highest anti-proliferation activity in MCF7, HeLa and HCT116 cells, which was much more effective than GA and the developing Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin). Meanwhile, compound 1b exhibited weaker growth inhibition effect on BRL cell line than GA and 17-AAG. 1b induced cell cycle arrest at the G2/M phase in MCF7 cells. Cleavage of PARP associated with apoptosis and degradation of the Hsp90 client protein Akt and Her2 was also induced by treatment of 1b in HeLa and MCF7 cell lines. In spite of the relatively weaker activity of 1b compared with GA and 17-AAG against HepG2 cells, 1b was further identified with lower hepatotoxicity than GA in vivo. CONCLUSION: Compound 1b is regarded as a new potential Hsp90 inhibitor with low hepatotoxicity for further study.

Specialised emission pattern of leaf trace in a late Permian (253 million-years old) conifer.

Leaf traces are important structures in higher plants that connect leaves and the stem vascular system. The anatomy and emission pattern of leaf traces are well studied in extant vascular plants, but remain poorly understood in fossil lineages. We quantitatively analysed the leaf traces in the late Permian conifer Ningxiaites specialis from Northwest China based on serial sections through pith, primary and secondary xylems. A complete leaf traces emission pattern of a conifer is presented for the first time from the late Palaeozoic. Three to five monarch leaf traces are grouped in clusters, arranged in a helical phyllotaxis. The leaf traces in each cluster can be divided into upper, middle and lower portions, and initiate at the pith periphery and cross the wood horizontally. The upper leaf trace increases its diameter during the first growth increment and then diminishes completely, which indicates leaf abscission at the end of the first year. The middle trace immediately bifurcates once or twice to form two or three vascular bundles. The lower trace persists as a single bundle during its entire length. The intricate leaf trace dynamics indicates this fossil plant had a novel evolutionary habit by promoting photosynthetic capability for the matured plant.

Design, synthesis and in vitro and in vivo antitumour activity of 3-benzylideneindolin-2-one derivatives, a novel class of small-molecule inhibitors of the MDM2-p53 interaction.

A novel class of small-molecule inhibitors of MDM2-p53 interaction with a (E)-3-benzylideneindolin-2-one scaffold was identified using an integrated virtual screening strategy that combined both pharmacophore- and structure-based approaches. The hit optimisation identified several compounds with more potent activity than the hit compound and the positive drug nutlin-3a, especially compound 1b, which exhibited both the highest binding affinity to MDM2 (Ki = 0.093 µM) and the most potent antiproliferative activity against HCT116 (wild type p53) cells (GI50 = 13.42 µM). Additionally, 1b dose-dependently inhibited tumour growth in BALB/c mice bearing CT26 colon carcinoma, with no visible sign of toxicity. In summary, compound 1b represents a novel and promising lead structure for the development of anticancer drugs as MDM2-p53 interaction disruptors.

Design, synthesis and antiproliferative activity of a novel class of indole-2-carboxylate derivatives.

Based on the chemical structure of Pyrroloquinoline quinone (PQQ), a novel class of indole-2-carboxylate derivatives was designed, synthesized and assayed for antiproliferative activity in cancer cells in vitro. The biological results showed that some derivatives exhibited significant antiproliferative activity against HepG2, A549 and MCF7 cells. Notably, the novel compounds, methyl 6-amino-4-cyclohexylmethoxy-1H-indole-2-carboxylate (6e) and methyl 4-isopropoxy-6-methoxy-1H-indole-2-carboxylate (9l) exhibited more potent antiproliferative activity than the reference drugs PQQ and etoposide in vitro, with IC50 values ranging from 3.78 ± 0.58 to 24.08 ± 1.76 µM. Further biological assay showed that both compounds 6e and 9l increased ROS generation dose-dependently, and induced PARP cleavage in A549 cells. Consequently, 6e and 9l appeared as promising anticancer lead compounds for further optimization.

Cardiac glycosides induce autophagy in human non-small cell lung cancer cells through regulation of dual signaling pathways.

Na(+)/K(+)-ATPase targeted cancer therapy has attracted increasing interests of oncologists in lung cancer field. Although multiple anti-cancer mechanisms of cardiac glycosides as Na(+)/K(+)-ATPase inhibitors are revealed, the role of autophagy and related molecular signaling pathway for the class of compounds in human non-small cell lung cancer (NSCLC) cells has not been systematically examined. We herein investigated the anti-cancer effects of two representative cardiac glycosides, digoxin and ouabain, in A549 and H460 cell lines. Both agents caused significant growth inhibition at nanomolar level. The cardiac glycosides were found to induce moderate G(2)/M arrest but not apoptosis at IC(50) level in the NSCLC cell lines. Moreover, autophagy was markedly induced by both agents, as evidenced by the time- and dose-dependent increase of LC3-II, up-regulation of Atg5 and Beclin1, as well as by the observations through acridine orange staining, transmission electron microscopy and quantification of GFP-LC3 fluorescence. Importantly, AMP-activated protein kinase (AMPK) pathway was activated, resulting in mammalian target of rapamycin (mTOR) deactivation during autophagy induction. Moreover, extracellular-signal-regulated kinase 1/2 (ERK1/2) activation was simultaneously found to be involved in the autophagy regulation. Co-treatment with respective inhibitors or siRNAs could either block the autophagic phenotypes and signals, or significantly increase the cellular viability, indicating the drugs-induced autophagy plays tumor-suppressing role. This work provides first evidence showing that the cardiac glycosides induce autophagy in human NSCLC cells through regulation of both mTOR and ERK1/2 signaling pathways. The autophagy may at least partially account for the growth inhibitory effects of the compounds in human NSCLC cells.