Immunomodulatory effect of Ganoderma lucidum polysaccharide extract on peritoneal macrophage function of BALB/c mice.

Ganoderma lucidum has been suggested as a natural immunomodulator. It is not yet clear exactly which combination of this extract is responsible for its immunomodulatory effects. Still, it appears that the 3-complement (CR3) receptor on the surface of immune cells acts as a receptor for beta-glucans (glucan-8), which is the main component of this extract. Since glucose-6-phosphate dehydrogenase (G6PD) plays a vital role in regulating macrophage functions, including nitric oxide production, we considered the effect of this extract on viability, G6PD enzyme activity, and nitric oxide (NO) production in peritoneal BALB/c macrophages. First, peritoneal macrophages of BALB/c mice were isolated and treated with concentrations (0.001, 0.01, 0.1, 1, 10, and 100 g/ml) of Ganoderma lucidum polysaccharide extract (GL-PS). After 24 hours of incubation by MTT test, we evaluated the viability of macrophages, and the effective dose was determined to be 0.1g/ml. To determine the specific activity of glucose-6-phosphates, they were incubated with GI-PS for 24 hours at a 0.1 mg/ml dose. Determination of protein concentration was obtained by the Bradford method in cell supernatant extract. Also, after 18 hours of incubation, the amount of nitric oxide (NO) production was measured using Grace colorimetric method. According to the results, a dose of 0.1µg/ml of Ganoderma lucidum polysaccharide extract had the most significant effect on the viability (stimulation coefficient) of peritoneal macrophages compared to other amounts (p <0.05). It was also found that a dose of 0.1µg/ml GL-PS increases NO production and the specific activity of the G6PD enzyme (p <0.05). Ganoderma lucidum, a medicinal fungus, is widely used in East Asian countries, especially in China, to increase the quality of life and longevity. After this study, we concluded that GL-PS extract has an immunomodulatory effect on macrophage activity. Therefore, the polysaccharide extract of this fungus can be used as a strengthening agent of the phagocytic system against infectious agents and pathogens such as the Leishmania parasite because of the production of nitric oxide by macrophages plays an essential role in defense against them.

Design, synthesis, cytotoxicities and DNA cleavage activities of dibenzoxepine and isoquinoline derivatives starting from dehydroabietylamine.

A series of novel hexahydrodibenzoxepine and quinazoline derivatives were designed and synthesized starting from dehydroabietylamine. The cytotoxicities of the compounds against L02 and HepG2 cell lines were investigated. Meanwhile, the plasmid DNA (Escherichia coli) cleavage of several heterocyclic derivatives was studied. These compounds exhibit remarkable activities on plasmid DNA pBR322. Our study provides useful information for developing new and more potent antitumor agents.

Distinctive Effects of Cytochalasin B in Chick Primary Myoblasts and Fibroblasts.

Actin-based structures play fundamental roles in cellular functions. However it remains controversial how cells cope with the absence of F-actin structures. This report focuses on short- and long-term effects of cytochalasin B (CB) on actin-complexes in fibroblasts and myoblasts. Thirty min of CB treatment dispersed subplasma actin cortices, lamellipodia, ruffled membranes, stress fibers and adhesion plaques into actin patches in fibroblasts and muscle cells. In contrast, 72 hrs CB treatment showed distinct morphological effects. Fibroblasts became giant multinucleated-finger shaped with 5 to 10 protrusions, 3-8 µm in width, and >200 µm in length. They lacked cortical actin, stress fibers, adhesion plaques and ruffled membranes but contained immense lamelliopodia with abnormal adhesion plaque protein complexes. Muscle cells transformed into multinucleated globular-shaped but contained normal I-Z-I and A-bands, indicating that CB did not interfere with the assembly of myofibrils. Within 30 min after CB removal, finger-shaped fibroblasts returned to their original shape and actin-containing structures rapidly reappeared, whereas muscle cells respond slowly to form elongated myotubes following CB washout. The capacity to grow, complete several nuclear cycles, assemble intermediate filaments and microtubules without a morphologically recognizable actin cytoskeleton raises interesting issues related to the role of the actin compartments in eukaryotic cells.

Discovering new agents active against methicillin-resistant Staphylococcus aureus with ligand-based approaches.

To discover new agents active against methicillin-resistant Staphylococcus aureus (MRSA), in silico models derived from 5451 cell-based anti-MRSA assay data were developed using four machine learning methods, including naïve Bayesian, support vector machine (SVM), recursive partitioning (RP), and k-nearest neighbors (kNN). A total of 876 models have been constructed based on physicochemical descriptors and fingerprints. The overall predictive accuracies of the best models exceeded 80% for both training and test sets. The best model was employed for the virtual screening of anti-MRSA compounds, which were then validated by a cell-based assay using the broth microdilution method with three types of highly resistant MRSA strains (ST239, ST5, and 252). A total of 12 new anti-MRSA agents were confirmed, which had MIC values ranging from 4 to 64 mg/L. This work proves the capacity of combined multiple ligand-based approaches for the discovery of new agents active against MRSA with cell-based assays. We think this work may inspire other lead identification processes when cell-based assay data are available.

Antitumor and scavenging radicals activities of some polyphenols related to dehydroabietylamine derivatives.

A novel series of polyphenols 4-9 were synthesized by the reaction of catechol with dehydroabietylamine derivatives. The antitumor activities of these compounds against L02 and HepG2 cells were investigated. Among them, compounds 4, 5, and 9 can inhibit HepG2 cells viability, but have lower inhibitory effect on L02 cells in the same concentration, indicating their potential for further development. Meanwhile, the novel series of polyphenols exhibited stronger radical-scavenging activities than the control groups.

A comparative study of antitumor activities and DNA cleavage on a class of dehydroabietylamine derivatives.

A series of novel dehydroabietylamine derivatives containing tricyclic diterpene structures were synthesized. The antitumor activities of these compounds against L02, Hey-1B and HepG2 cells were investigated. Significant activity was discovered forfourteen analogs. Meanwhile these compounds exhibit DNA cleavage activities on plasmid DNA (Escherichia coli), which depend on the Schiff base structure and the substituent of the aromatic moiety. Our findings present further information on the relationship between the chemical structure, biological function and DNA cleavage characteristics.

N-Benzoyl-12-nitrodehydroabietylamine-7-one, a novel dehydroabietylamine derivative, induces apoptosis and inhibits proliferation in HepG2 cells.

The high biological activity of dehydroabietylamine derivatives has been reported previously. In this study, we aimed to screen 73 dehydroabietylamine derivatives as potential candidate inhibitors in liver cancer cells. Initially, the compounds structural activity relationship analysis was explored and N-benzoyl-12-nitrodehydroabietylamine-7-one (compound 81) was shown to have significant growth inhibitory activity in the human liver carcinoma cell line, HepG2. Further research into the anti-proliferative effect on HepG2 cells mediated by compound 81 was undertaken. The results suggest that compound 81 effectively induced apoptosis in HepG2 cells characterized by nuclear staining of DAPI, TUNEL assay and the activation of caspase-3. A decreased level of anti-apoptotic protein Bcl-2 and increased apoptotic Bax were also observed. Furthermore, Ki-67 protein staining and the BrdU incorporation assay showed that compound 81 significantly inhibited the proliferation of HepG2 cells. Cell cycle components analysis found that expression of cyclin D1 and cyclin B1 was reduced in HepG2 cells with compound 81 treatment, whereas the content of p21(Waf1/Cip1) was increased. Taken together, our data indicate that compound 81 induces apoptosis and inhibits proliferation in HepG2 cells, and may be a promising candidate in the development of a novel class of antitumor agents.

Synthesis and NMR spectral studies of the 7-C60-adduct of n,n-(tetrachlorophthaloyl) dehydroabietylamine.

The 7-C60-adduct of N,N-(tetrachlorophthaloyl)dehydroabietylamine was synthesized for the first time and characterized by IR, UV-vis, mass and NMR spectral studies. The ¹H-NMR and ¹³C-NMR resonance signals of the new compound are unambiguously assigned by using homo- and heteronuclear 2D NMR spectroscopic techniques such as COSY, ROESY, HSQC and HMBC. The C1 symmetric structure with 6,6-junction of compound was determined.

Synthesis and antitumour activities of a novel class of dehydroabietylamine derivatives.

Structural modification is still a popular and important route in the forest chemical field for finding novel tricyclic diterpenes with more potential bioactivities and broad bioactive spectra. In this study, a series of dehydroabietylamine derivatives containing tricyclic diterpene structures were synthesised through oxidation in the 7th position of ring B and nitrification in the 12th position of ring C using dehydroabietylamine as the starting material. Structures of the synthesised compounds were confirmed by IR, (1)H-NMR, (13)C-NMR, MS and HRMS. The cytotoxicities of these compounds against PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells by the MTT assay were investigated. The results showed that the presence of a nitro group at 12th position and a carbonyl group at 7th position resulted in an increase of cytotoxic activity. Our findings present more evidence, showing the relationship between the chemical structure and biological function.

Effective asymmetry in gap junctional intercellular communication between populations of human normal lung fibroblasts and lung carcinoma cells.

The dysfunction of homologous and/or heterologous gap junctional intercellular communication (GJIC) has been implicated in tumorigenesis of many kinds of cells. Here we have characterized GJIC and the expression of connexins in six human lung carcinoma cell lines and normal lung fibroblasts (HLF). Compared with HLF, all the carcinoma cells showed reduced or little homologous GJIC. They expressed remarkably reduced connexin(Cx)43 mRNA and variable levels of Cx45 mRNA, but neither Cx43 nor Cx45 protein could be detected. However, using a preloading assay, transfer of calcein was observed between donor HLF cells and first order neighboring recipient tumor cells (recipient cells in 1000-fold excess). Transfer from tumor to HLF cells under the same conditions was not seen, although increasing the ratio of donor tumor cells to recipient HLF cells and plating the cells at low density did reveal weak transfer from tumor cells to HLF. Transfection of Cx43 into giant cell carcinoma PG cells increased homologous communication and eliminated the rectifying behavior of heterologous communication. This indicates that the apparent rectification of dye transfer between normal and tumor cells was a product of low rates of heterologous transfer linked to (i) rapid dilution of the dye to below detectable limits through a very well coupled cell population (tumor to HLF) and (ii) concentration of dye in immediate neighbors in a poorly coupled cell population (HLF to tumor cells). These results suggest that the coupling levels may need to exceed a certain threshold to allow propagation of signals over a sufficient distance to affect behavior of a cell population. We propose that the relative rates of heterologous and homologous coupling of cell populations and the 'pool size' of shared metabolites in tumor cells and the surrounding normal tissue are likely to be very important in the regulation of their growth.