Synthesis of thioether andrographolide derivatives and their inhibitory effect against cancer cells.

A series of novel thioether andrographolide derivatives were synthesized by incorporating various aromatic (or heteroaromatic) substituents into C-12 or 14-OH. A total of 38 andrographolide derivatives were prepared and evaluated for their in vitro inhibitory activity against cancer cells. All the derivatives exhibited better activity against prostate cancer cells (PC-3) than the parent compound. Among these, compounds 6a, 8, 9, 17, 19, 31, and 32 demonstrated good activity. These compounds were further evaluated for their anticancer activities against other cancer cell lines including MCF-7, MDA-MB-231, and A549. Compounds 31 and 32 showed excellent activity against MCF-7 with an IC50 value of 0.7 and 0.6 µM, respectively. The absolute configuration of 15a was determined via single-crystal X-ray diffraction. The activity of 6a (12S), which was the precursor of 15a, was better than that of the diastereoisomer 6b (12R). Moreover, the preliminary structure-activity relationship has been summarized. The results obtained herein are very important for further optimization of andrographolide.

Meta-Analysis of the Association between Tea Intake and the Risk of Cognitive Disorders.

BACKGROUND: Alzheimer's disease is a common neurodegenerative disorder in elderly. This study was aimed to systematically evaluate the association between tea intake and the risk of cognitive disorders by meta-analysis. METHODS AND FINDINGS: PubMed, Embase and Wanfang databases were systematically searched and a total of 26 observational studies were included in this study. Odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were calculated and pooled by using fixed or random effects models according to the degree of heterogeneity. RESULTS: The overall pooled analysis indicated that tea intake could significantly reduce the risk of cognitive disorders (OR = 0.65, 95%CI = 0.58-0.73). Subgroup analyses were conducted based on study design, population, frequency of tea drinking and type of cognitive disorders. The results showed that tea drinking was significantly associated with the reduced incidence of cognitive disorders in all of subgroups based on study design and frequency of tea drinking. In particular, tea drinking was inversely associated with the risk of cognitive impairment (CoI), mild cognitive impairment (MCI), cognitive decline and ungrouped cognitive disorders. Moreover, for population subgroups, the significant association was only found in Chinese people. CONCLUSION: Our study suggests that daily tea drinking is associated with decreased risk of CoI, MCI and cognitive decline in the elderly. However, the association between tea intake and Alzheimer's disease remains elusive.

Significantly inhibitory effects of low molecular weight heparin (Fraxiparine) on the motility of lung cancer cells and its related mechanism.

Low molecular weight heparin (LMWH) improving the cancer survival has been attracting attention for many years. Our previous study found that LMWH (Fraxiparine) strongly downregulated the invasive, migratory, and adhesive ability of human lung adenocarcinoma A549 cells. Here, we aimed to further identify the antitumor effects and possible mechanisms of Fraxiparine on A549 cells and human highly metastatic lung cancer 95D cells. The ability of cell invasion, migration, and adhesion were measured by Transwell, Millicell, and MTT assays. FITC-labeled phalloidin was used to detect F-actin bundles in cells. Chemotactic migration was analyzed in a modified Transwell assay. Measurement of protein expression and phosphorylation activity of PI3K, Akt, and mTOR was performed with Western blot. Our studies found that Fraxiparine significantly inhibited the invasive, migratory, and adhesive characteristics of A549 and 95D cells after 24 h incubation and showed a dose-dependent manner. Fraxiparine influenced the actin cytoskeleton rearrangement of A549 and 95D cells by preventing F-actin polymerization. Moreover, Fraxiparine could significantly inhibit CXCL12-mediated chemotactic migration of A549 and 95D cells in a concentration-dependent manner. Furthermore, Fraxiparine might destroy the interaction between CXCL12-CXCR4 axis, then suppress the PI3K-Akt-mTOR signaling pathway in lung cancer cells. For the first time, our data indicated that Fraxiparine could significantly inhibit the motility of lung cancer cells by restraining the actin cytoskeleton reorganization, and its related mechanism might be through inhibiting PI3K-Akt-mTOR signaling pathway mediated by CXCL12-CXCR4 axis. Therefore, Fraxiparine would be a potential drug for lung cancer metastasis therapy.

Cationic lipids containing protonated cyclen and different hydrophobic groups linked by uracil-PNA monomer: synthesis and application for gene delivery.

In this report, as candidates for non-viral gene vectors, cationic lipids L1, L2 and L3 based on protonated cyclen and different hydrophobic groups (cholesterol, dodecanol or diosgenin) linked by PNA monomer were designed and synthesized. Their liposomes were easily prepared by mixing the synthesized lipids with dioleoylphosphatidyl ethanolamine (DOPE) under appropriate mole ratios. Agarose gel retardation and fluorescent titration by ethidium bromide (EB) showed the strong DNA-binding ability with the K(sv) values of 1.21 × 10(7), 3.76 × 10(6) and 2.90 × 10(6) M(-1) for the liposomes formed from L1-L3, respectively. These liposomes could retard pDNA at an N/P ratio of 3 and form lipoplexes with sizes around 200-300 nm and zeta-potential values of +20-50 mV at N/P ratio from 4 to 10. Besides, the cytotoxicity of the three lipoplexes assayed by MTT is quite different. The results from in vitro transfection in HEK 293T and A549 cell lines showed that the transfection efficiency of L3/DOPE/DNA lipoplex at an N/P ratio of 6 and lipid/DOPE mole ratio of 1:2 is slightly higher than that of Lipofectamine 2000™, indicating that the title PNA monomer-based cationic lipids have great potential to be efficient non-viral gene vector.

Heteromerization and colocalization of TrpV1 and TrpV2 in mammalian cell lines and rat dorsal root ganglia.

Coassociation of the vanilloid transient receptor potential (Trp) ion channels, TrpV1 and TrpV2, was investigated by immunoprecipitation and immunofluorescence in transfected mammalian cell lines, rat dorsal root ganglia and spinal cord. TrpV1/TrpV2 heteromeric complexes were coimmunoprecipitated from human embryonic kidney cells and F-11 dorsal root ganglion hybridoma cells following their transient coexpression. Immunofluorescent labelling of transfected F-11 cells revealed colocalization of TrpV1 and TrpV2 at the cell surface. Immunoprecipitation from rat dorsal root ganglion lysates identified a minor population of receptor complexes composed of TrpV1/TrpV2 heteromers, consistent with a small proportion of cells double-labelled with TrpV1 and TrpV2 antibodies in rat dorsal root ganglion sections. TrpV1/TrpV2 receptor complexes may represent a functionally distinct ion channel complex that may increase the diversity observed within the Trp ion channel family.

Modulation of brain stem monoamines and gamma-aminobutyric acid by NK1 receptors in rats.

To understand the role of substance P in stress, anxiety and depression, we have investigated in rats the relationship between NK1 receptors and monoamines or GABA, and between substance P and serotonin (5-HT) in brain stem neurons by immunohistochemical double-staining techniques. In the periaqueductal gray and dorsal raphe nucleus, there was no colocalization between NK1 and 5-HT or between NK1 and tyrosine hydroxylase (a marker for adrenaline and dopamine neurons). However, many GABA-positive neurons (> 50%) were NK1 positive, and some substance P-positive neurons were 5-HT positive as well. Almost all locus coeruleus noradrenaline neurons were NK1 positive. Therefore, substance P may promote stress by activating noradrenaline neurons directly and inhibiting 5-HT neurons indirectly via GABA neurons.

Cholera toxin B subunit labeling in lamina II of spinal cord dorsal horn following chronic inflammation in rats.

We have investigated the effect of inflammation on the labeling pattern of cholera toxin B subunit (CTB)-conjugated horseradish peroxidase, an A-fiber marker, by an intra-sciatic nerve injection of the tracer. Following chronic inflammation in one hind paw in rats, there was substantial CTB labeling in lamina II of the spinal dorsal horn, which is normally absent. However, there was no change in the labeling pattern of wheat germ agglutinin or fluoride resistant acid phosphatase/thiamine monophosphatase, two C-fiber markers. The CTB labeling in lamina II after peripheral nerve injury has been interpreted as central sprouting of A-fibers or uptake of the tracer by injured C-fibers. Our results suggest that chronic inflammation and nerve injury may share some common mechanisms in generating allodynia and hyperalgesia.

Expression of capsaicin receptor (VR1) by myelinated primary afferent neurons in rats.

The expression of capsaicin (VR1) receptor by A-fiber primary afferent neurons has been investigated by double immunohistochemical staining with VR1 and 200 kD neurofilament (NF200; an A-fiber marker) antibodies, and by VR1 immunohistochemical staining in combination with cholera toxin B subunit (CTB; also an A-fiber marker) retrograde tracing. Approximately 30% of the VR1-positive dorsal root ganglion (DRG) neurons were NF200-positive. Intra-sciatic nerve injection of CTB labeled over 30% of the VR1-positive neurons in the L5 DRG. Size frequency distribution analysis revealed that these VR1 and NF200, or VR1 and CTB, double-labeled neurons were predominantly small and medium sized. These results suggest that capsaicin receptors are likely to be expressed by Adelta-fiber neurons as well as C-fiber neurons.

Morphine, the NMDA receptor antagonist MK801 and the tachykinin NK1 receptor antagonist RP67580 attenuate the development of inflammation-induced progressive tactile hypersensitivity.

Normally-innocuous low-intensity tactile stimuli applied to inflamed tissue induce a progressive decrease in the mechanical flexion withdrawal threshold, the phenomenon of progressive tactile hypersensitivity (PTH). The effects of the mu opioid receptor agonist morphine, the non-competitive NMDA receptor antagonist MK801 and the tachykinin NK1 receptor antagonist RP67580 on the development and maintenance of PTH has now been investigated behaviourally in rats inflamed 48 h earlier by intraplantar complete Freund's adjuvant injection. A standard protocol of eight light tactile stimuli applied to the dorsum of the inflamed paw every 4 s at 5 min intervals resulted, over 60 min, in a 70% fall in mechanical threshold from the pre-conditioning baseline value. Morphine administered before the tactile stimuli at 0.05 mg/kg i.p. had no effect on either baseline thresholds or PTH. At 0.5 mg/kg, morphine prevented the establishment of PTH without changing baseline thresholds. At 5 mg/kg morphine produced analgesia, increasing thresholds above the baseline. MK801 pre-treatment at 0.01 and 0.001 mg/kg i.p. significantly attenuated the development of progressive tactile hyperalgesia without an effect on basal thresholds. RP67580 pre-treatment at 0.1 mg/kg i.p. had no effect, but at both I and 10 mg/kg, attenuated progressive tactile hypersensitivity without changing baseline values. To test the effect of the drugs on established PTH, they were administered 90 min after the commencement of intermittent tactile stimulation to the inflamed hindpaw, when thresholds had reached a plateau. Morphine (0.5 mg/kg) and MK801 (0.01 mg/kg) produced only a small reduction in sensitivity and RP67580 (1 mg/kg) had no effect. These results suggest that the induction of inflammatory progressive tactile hypersensitivity is sensitive to morphine, and to a lesser extent NMDA and NKI receptor antagonists, but these compounds at a dose that do not alter baseline values, do not normalise established tactile hypersensitivity.

Progressive tactile hypersensitivity: an inflammation-induced incremental increase in the excitability of the spinal cord.

Two established phenomena contribute to the generation of post-injury pain hypersensitivity: peripheral sensitization, an increase in transduction sensitivity of high threshold A delta and C-fibre nociceptors, and central sensitization, an increase in excitability of neurones in the spinal cord triggered exclusively by C-fibre inputs. We now describe a novel phenomenon: progressive tactile hypersensitivity, which contributes to a cumulative allodynia during inflammation. Behavioural measurements in conscious intact animals showed that repeated light touch stimuli delivered at 5-min intervals to an inflamed paw, established 48 h earlier by an intra-plantar injection of complete Freund's adjuvant (CFA), resulted in a progressive reduction in the mechanical withdrawal threshold by more than 75%, from its already hypersensitive basal level. This hypersensitive state persisted for several hours after discontinuing the touch stimuli and did not occur in non-inflamed animals. To monitor nociceptive processing and the afferent fibres responsible, we also measured activity in posterior biceps femoris/semitendinosus flexor motor neurones. In non-inflamed decerebrate-spinal rats, the cutaneous mechanical threshold and pinch-evoked activity of these neurones are stable when tested repeatedly at 5-min intervals and are characterised by absent or small responses to low intensity mechanical stimuli or electrical activation of A beta-fibres. In inflamed animals, the spontaneous activity, touch-, pinch- and A beta-afferent-evoked responses of hamstring flexor motor neurones are significantly increased. The flexor reflex becomes, moreover, progressively more sensitized by repetition every 5 min, of standard mechanical stimuli (touch and pinch), that do not modify excitability in control non-inflamed animals. A cumulative increase in A beta-afferent-evoked responses also occurs when the test stimulus only comprises stimulation of the sural nerve at A beta strength (10 Hz, 10 sec), showing that A beta-afferents have the capacity to produce progressive hypersensitivity. Progressive hypersensitivity, measured here as a progressive tactile allodynia after inflammation in either intact or decerebrate-spinal rats, with its gradual build-up and contribution from A beta fibres, is very different from the central sensitization induced by C-fibre stimulation which is characterised by a peak increase in excitability soon after the conditioning input followed by a steady decrement to baseline levels. Progressive hypersensitivity is likely to be the consequence of an alteration in the function and phenotype of afferents innervating inflamed tissue and the pattern of excitation they produce in spinal neurones. The phenomenon may have an important role in the development of inflammatory pain and hypersensitivity.

Noxious stimuli induce an N-methyl-D-aspartate receptor-dependent hypersensitivity of the flexion withdrawal reflex to touch: implications for the treatment of mechanical allodynia.

Primary C-fiber afferents can induce a state of increased excitability in spinal cord neurons amplifying their responsiveness to noxious and innocuous stimuli--the phenomenon of central sensitization. We have examined whether the hypersensitivity to low-intensity stimuli (mechanical allodynia) evoked by C-afferent conditioning inputs is NMDA receptor dependent. The enhancement by C-afferent conditioning stimuli of the normally low or absent cutaneous touch-evoked responses of posterior biceps femoris/semitendinosus flexor motoneurons in the decerebrate-spinal rat has been used as a model of touch-evoked allodynia. Three different noxious conditioning stimuli (electrical stimulation of the sural nerve at C-fiber strength, cutaneous application of mustard oil and the intramuscular (i.m.) injection of bradykinin) enhanced touch-evoked responses and decreased the threshold of the flexor reflex to mechanical stimulation. Intrathecal (i.t.) injections of NMDA (20 pmol) produced a similar effect. A non-competitive NMDA antagonist MK 801, administered prior to the conditioning input at doses that did not modify the baseline reflex, significantly attenuated both the enhancement of touch-evoked responses and the threshold decrease following the noxious conditioning stimuli. Treatment with MK 801 some time after the conditioning input when the mechanical hypersensitivity is fully established, also reduced the increased touch-evoked responses. The reduction in threshold and the increase in touch responsiveness induced by cutaneous and muscle noxious C-fiber conditioning stimuli in the rat spinal cord are, therefore, both prevented and reversed by NMDA receptor antagonism. NMDA antagonists may be potentially useful, therefore, in treating postinjury pain hypersensitivity.