Enhancement of Dissolving Capacity and Reducing Gastric Mucosa Irritation by Complex Formation of Resibufogenin with ß-Cyclodextrin or 2-Hydroxypropyl-ß-cyclodextrin.

Resibufogenin (RBG) is a natural medicinal ingredient with promising cardiac protection and antitumor activity. However, poor solubility and severe gastric mucosa irritation restrict its application in the pharmaceutical field. In this study, the inclusion complex of RBG with ß-cyclodextrin (ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was prepared using the co-evaporation method, and the molar ratio of RBG to CD was determined to be approximately 1:2 by continuous variation plot for both CDs. The formation of inclusion complexes between RBG and each CD (RBG/ß-CD and RBG/HP-ß-CD) was evaluated by phase solubility study, Fourier transform infrared spectroscopy, and thin-layer chromatography. Powder X-ray diffraction and differential scanning calorimetry confirmed drug amorphization and encapsulation in the molecular cage for both CDs. Moreover, the inclusion complexes' morphologies were observed using scanning electron microscopy. The dissolution rate of the inclusion complexes was markedly improved compared to that of RBG, and the complexes retained their antitumor activity, as shown in the in vitro cytotoxicity assay on a human lung adenocarcinoma cancer (A549) cell line. Moreover, less gastric mucosal irritation was observed for the inclusion complex. Thus, the inclusion complex should be considered a promising strategy for the delivery of poorly water-soluble anticancer agents, such as RBG.

Two New Indole Alkaloids from Toad Venom of Bufo bufo gargarizans.

Two new indole alkaloids, Bufotenidine B (2) and Bufocarboline A (6), along with seven known indole alkaloids (1, 3-5, and 7-9) and three organic acids (10-12), were isolated from the water extract of toad venom. The structures of the new alkaloids were elucidated by extensive spectroscopic methods. The absolute configurations of 4, 6, and 8 were determined for the first time by electronic circular dichroism (ECD) calculations. The cytotoxic activity of all compounds was tested against human malignant melanoma cells A375 by the MTT method, and no antitumor activity was observed.

A new indole alkaloid from the traditional Chinese medicine Chansu.

A new indole alkaloid N'-formylserotonin (1), along with five known indole alkaloids N'-methylserotonin (2), 5-hydroxy-1H-indole-3-carbaldehyde (3), N-acetylserotonin (4), 6-hydroxy-1-oxo-3,4-dihydro-ß-carboline (5), and bufoserotoin C (6), were isolated from the water extract of traditional Chinese medicine Chansu. Their structures were elucidated on the basis of spectral analyses. The cytotoxicities of 1-6 against human lung adenocarcinoma epithelial cells A549 were tested using the MTT method. Compound 6 exhibited stronger cytotoxic effect than 5-FU, and 1-5 showed no cytotoxic effects. Bufoserotonin C is one of the cytotoxic components in water-soluble extract of Chansu.

Gelatin promotes murine fibrosarcoma L929 cell detachment and protects the cells from TNFα-induced cytotoxicity.

PURPOSE: Gelatin has been considered to exist as intermediate substance of collagen catabolism in tissue remodeling or under inflammatory conditions. We have initiated the study on possible biological functions of gelatin that can exist temporally and locally under the conditions of remodeling and inflammation Materials and methods: To this purpose, we investigated cell proliferation and survival on gelatin-coated dishes and the response to tumor necrosis factor α (TNFα)-induced cytotoxicity in L929 cells. Autophagy level, ATP level, and ROS generation are examined. RESULTS: L929 cells detached from the gelatin-coated dishes and formed multicellular aggregates. TNFα-induced cytotoxicity in L929 cells was inhibited by gelatin-coating culture. The cells on gelatin-coated dishes showed reduced cellular ATP levels and increased adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation, leading to increased ROS generation and autophagy. CONCLUSION: This study showed that gelatin-coated culture protected L929 cells from TNFα-induced cytotoxicity and suggested for a possible pathophysiological function of gelatin in regulating cellular functions.

A New Indole Alkaloid from the Toad Venom of Bufo bufo gargarizans.

A new indole alkaloid named bufobutarginine (1), along with three known bufotenines, namely, serotonin (2), bufotenidine (3), and bufotenine (4), were isolated from the water extract of toad venom. Their structures were elucidated by spectral methods. This is the first time that arginine has been found to be involved in the biosynthesis of bufotenines in parotid of toad. The cytotoxic activities of these compounds have been assayed against A375 and A549 cell lines by the MTT method; however, they showed no cytotoxic activities.

[Cloning, prokaryotic expression and function of the Bufo bufo gargarizans tryptophan hydroxylase I gene].

Tryptophan hydroxylase I（TPH1） catalases the 5-hydroxylation reaction of L-Trp, which is the rate-limiting step in the synthesis of serotonin. Serotonin, a major component of Bufonis venenum, is involved in numerous physiological functions as an important neurotransmitter. In this study, BbgTPH1 cDNA was cloned from the parotid gland of Bufo bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pMAL-BbgTPH1, and the induced conditions to express the recombinant BbgTPH1 in E. coli TB1 cells were optimized. The full length of BbgTPH1 is 1 984 bp (GenBank accession No. JQ768313) with a 1 443 bp open reading frame (ORF) encoding a 480 amino acid residues. The deduced protein molecular weight is 55.2 k Da and its theoretical isoelectric point is 5.58. The sequence includes conserved domain and special signal sequence of the aromatic amino acid hydroxylase (AAAH) superfamily. Homologous alignment showed that BbgTPH1 shared a high homology with other species. Phylogenetic tree showed the closest relative to BbgTPH1 was Xenopus tropical-TPH1. The best induction conditions of recombinant BbgTPH1 were 0.5 mmol·L(-1) IPTG at 20 ℃ for 8 h. The function of BbgTPH1 was identified by in vitro enzymatic reaction and the recombinant BbgTPH1 was able to produce 5-hydroxytryptophan by catalyzing tryptophan. This study represents the first time of cloning and identification of the function of TPH1 in Bufo genus. The results of this study will be an important foundation for future studies of biosynthesis of bufotenines in the parotid gland of B. bufo gargarizans.

Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death.

Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death.

Anthraquinone metabolites isolated from the rhizosphere soil Streptomyces of Panax notoginseng (Burk.) F. H. Chen target MMP2 to inhibit cancer cell migration.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng (Burk.) F. H. Chen belongs to the Araliaceae family. It has been used by traditional Chinese people in Northeast Asia for centuries as an antidiabetic, antioxidant, antitumor agent, etc. Endophytic or rhizospheric microorganisms play key roles in plant defense mechanisms, and they are essential in the discovery of pharmaceuticals and valuable new secondary metabolites. In particular, endophytic or rhizospheric microorganisms of traditional medicinal plants. AIM OF THE STUDY: To discover valuable new secondary metabolites from rhizosphere soil Streptomyces sp. SYP-A7185 of P. notoginseng, and to explore potential bioactivities and targets of metabolites protrusive function. MATERIALS AND METHODS: The metabolites were obtained via column chromatography and identified by multiple spectroscopic analyses. The antitumor, antioxidant, antibacterial, and antiglycosidases effects of isolated metabolites were tested using 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetazolium bromide (MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 96-well turbidimetric, and α-glucosidase inhibitory assays. The potential antitumor targets were predicted through network pharmacological approaches. The interactions between metabolites and target were verified by molecular docking and biolayer interferometry (BLI) assay. The effects of cancer cells migration were detected through wound healing assays in A549 and MCF-7. Other cellular validation experiments including reverse transcription-quantitative PCR (RT‒qPCR) and western blotting (WB) were used to confirm the hypothesis of network pharmacology. RESULTS: Five different chemotypes of anthraquinone derivatives (1-10), including six new compounds (3, 6-10), were identified from Streptomyces sp. SYP-A7185. Compounds 1-6 and 9 displayed moderate to strong cytotoxicity on five human cancer cell lines (A549, HepG2, MCF-7, MDA-MD-231, and MGC-803). Moreover, matrix metalloproteinase-2 (MMP2) were predicted as a potential antitumor target of metabolites 1-6 and 9 by comprehensive network pharmacology analysis. Later, BLI assays revealed strong intermolecular interactions between MMP2 and antitumor metabolites, and molecular docking results showed the interaction of metabolites 1-6 and 9 with MMP2 was dependent on the crucial amino acid residues of LEU-83, ALA-84, LEU-117, HIS-131, PRO-135, GLY-136, ALA-140, PRO-141, TYR-143, and THR-144. These results implied that metabolites (1-6 and 9) might inhibit cancer cell migration besides cancer cell proliferation. After that, the cell wound healing assay showed that the cell migration processes were also inhibited after the treatments of compounds 1 and 3 in A549 and MCF-7 cells. In addition, the RT‒qPCR and WB results demonstrated that the gene expression levels of MMP2 were decreased after the treatment with compounds 1 and 3 in A549 and MCF-7 cells. Besides, compound 2 displayed moderate antioxidant activity (EC50, 27.43 µM), compounds 3 and 6 exhibited moderate antibacterial activity, and compound 3 inhibited α-glucosidase with an IC50 value of 13.10 µM. CONCLUSIONS: Anthraquinone metabolites, from rhizosphere soil Streptomyces sp. of P. notoginseng, possess antitumor, antioxidant, antibacterial, and antiglycosidase activities. Moreover, metabolites 1 and 3 inhibit cancer cells migration through downregulating MMP2.

p38-NF-κB-promoted mitochondria-associated apoptosis and G2/M cell cycle arrest in norcantharidin-treated HeLa cells.

Previous study proved that norcantharidin (NCTD) could exert its anticancer activity in a variety of malignant cell lines, including human cervical carcinoma HeLa cells. In this study, we found that NCTD-activated p38 mitogen-activated protein kinase (p38 MAPK)-nuclear transcription factor kappa B (NF-κB) signaling pathway induced mitochondrial apoptotic pathway activation and G2/M cell cycle arrest in HeLa cells. NCTD-induced mitochondria-associated apoptosis was concomitant with the collapse of mitochondrial membrane potential (ΔΨ(m)), translocation of Bax, down-regulation of Bcl-2 expression, and release of cytochrome c. NCTD-led G2/M cell-cycle arrest was associated with the up-regulated p21 and p-cdc25c expression and the down-regulated cyclin B and cdc2 expression. Treatment of the cells with p38 inhibitor SB203580 and NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) showed that p38 functioned upstream of NF-κB, while augmented apoptosis and cell cycle arrest were induced in response to NCTD with NF-κB activation. Intriguingly, NF-κB had a negative feedback regulatory effect on p38 activation. Moreover, NCTD-induced apoptosis and cell cycle arrest were significantly blocked by SB203580 and PDTC but not by pifithrin-α (p53 inhibitor). Therefore, p38-NF-κB induced mitochondrial apoptotic pathway and G2/M cell cycle arrest in NCTD-treated HeLa cells.

Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways.

Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H(2)O(2)), superoxide dismutase (SOD) (scavenger of O(2)(•-)), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H(2)O(2) or O(2)(•-) showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.

Silibinin activated ROS-p38-NF-κB positive feedback and induced autophagic death in human fibrosarcoma HT1080 cells.

Our previous results demonstrated that silibinin induced autophagic and apoptotic cell death dependent on reactive oxygen species (ROS especially H(2)O(2) and [image omitted] ) in HT1080 cells. In this study, we further show that p38-NF-κB pathway is involved in silibinin-induced ROS-mediated autophagy. Cells were pretreated with serum-free media for 24 h before being treated with silibinin. Generation of ROS and autophagy was detected in 15 min and 1 h, respectively. Development of autophagy was supported by an upregulated expression of Beclin-1 and conversion of light chain (LC3-I-LC3-II). Expression of p38/p-p38 and transposition of NF-κB from cytoplasm to nuclei were also increased. Inhibitors of p38 and NF-κB and scavengers of H(2)O(2) and O(2)(*-) reduced both generation of ROS and simultaneous occurrence of silibinin-induced autophagy. Besides, expression of p38/p-p38 and transposition of NF-κB from cytoplasm to nuclei were decreased by these two ROS scavengers. ROS and p38-NF-κB pathway were possibly cooperated in a positive feedback mechanism. Inhibition of p38, NF-κB, H(2)O(2), or O(2)(*-) rescued cells from silibinin-induced death in a long-term (12 h) manner. According to the previous study that silibinin-induced autophagy was a positive regulator of apoptotic cell death, it was possible that ROS and p38-NF-κB mediated silibinin-induced autophagy and eventually led to cell death.

Cytotoxic and antimicrobial indole alkaloids from an endophytic fungus Chaetomium sp. SYP-F7950 of Panax notoginseng.

Two new compounds chetoseminudin F (1) and G (2) together with eleven known compounds were isolated from the solid fermentation products of the endophytic fungus Chaetomium sp. SYP-F7950. The structures of the isolated compounds were elucidated by extensive spectroscopic analyses, including 1D and 2D NMR, and HRFABMS experiments. The absolute configurations of chetoseminudin F (1) and G (2) were determined by comparing the electronic circular dichroism (ECD) spectrum with those of the reported references. A plausible biogenetic pathway for compounds 1-6 and 9-13 was proposed. These isolates were also evaluated for their antimicrobial and antitumor activity, revealing that chetoseminudin F (1) displayed more potent cytotoxicity against MDA-MB-231 cells with an IC50 value of 26.49 µmol L-1 more than the common chemotherapeutic agent (paclitaxel). In antimicrobial assay, compounds 6, 9, 11 and 12 had strong antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium and antifungal activity against Candida albicans with minimum inhibitory concentration (MIC) values ranging from 0.12 to 9.6 µg mL-1; meanwhile compounds 6, 8, 9 and 12 exhibited strong cytotoxicity with IC50 values of 2.75-8.68 µmol L-1 against tumor cell lines A549 and MDA-MB-231. In addition, morphological observation showed that treatment with compounds 6, 9 and 12 increased the mean length of B. subtilis by 1.6 to 1.8-fold. In silico molecular docking was applied to study the binding interactions between the compounds and the active sites of filamentous temperature-sensitive protein Z (FtsZ) from B. subtilis. Compounds 6, 9 and 12 displayed the low binding energies, strong H-bond interactions with FtsZ. On the basis of the antimicrobial activities, cellular phenotype observation and docking studies, compounds 6, 9 and 12 are considered to be a promising antimicrobial inhibitor of FtsZ.

Activation of Wnt/ß-catenin signaling by lithium chloride attenuates d-galactose-induced neurodegeneration in the auditory cortex of a rat model of aging.

Degeneration of the central auditory system, which is characterized by reduced understanding of speech and source localization of sounds, is an important cause of age-related hearing loss (presbycusis). Accumulating evidence has demonstrated that Wnt/ß-catenin signaling plays an essential role in the development of the auditory system but its potential role in presbycusis remains unclear. In this study, we used a rat model of aging, created by chronic systemic exposure to d-galactose (d-gal), and explored changes in Wnt/ß-catenin signaling in the auditory cortex. A decrease in Wnt/ß-catenin signaling in the auditory cortex was found in both naturally aging and d-gal-mimetic aging rats, as indicated by increased GSK3ß activity and decreased ß-catenin activity. Moreover, lithium chloride (Licl), an activator of Wnt signaling pathway, was administered long term to 15-month-old d-gal-treated rats. Activation of Wnt/ß-catenin signaling by Licl attenuated d-gal-induced auditory cortex apoptosis and neurodegeneration. Bmi1, a transcription factor implicated in antiaging and resistance to apoptosis, can be modulated by ß-catenin activity. Here, we showed that the expression of Bmi1 was reduced and the expression of its downstream genes, p16INK4a , p19Arf , and p53 were increased in the auditory cortex both of naturally aging and d-gal-mimetic aging rats. In addition, Licl significantly increased Bmi1 expression and reduced p16INK4a, p19Arf, and p53 expression. Our results indicated that decreased Wnt/ß-catenin signaling might participate in the pathogenesis of central presbycusis through modulating the expression of Bmi1. Wnt/ß-catenin signaling might be used as a potential therapeutic target against presbycusis.

Induction of protective autophagy against apoptosis in HepG2 cells by isoniazid independent of the p38 signaling pathway.

Isoniazid (INH), one of the first-line anti-tuberculosis drugs, is adversely associated with hepatotoxicity in the clinic. However, the detailed mechanism of this side effect is still unclear. The traditional theory that cytochrome P450 2E1 is involved in INH-induced hepatotoxicity remains controversial, therefore other mechanisms by which INH exerts hepatotoxicity need to be investigated. In the current study, we showed that in vitro treatment of human hepatocarcinoma HepG2 cells with INH induced caspase-dependent apoptosis through extrinsic and intrinsic pathways. It was characterized by the increased population of apoptotic cells using Annexin V/propidium iodide (PI) double staining by flow cytometry, and by the activation of caspases 8, 9, 3 and poly (ADP-ribose)-polymerase (PARP) proteins by western blotting. INH treatment also induced autophagy as shown by the upregulated levels of microtubule-associated protein 1 light chain 3-II (LC3-II), increased GFP-LC3 punctates, and elevated monodansylcadaverine (MDC) fluorescence intensity. The measurement of the autophagic flux using chloroquine (CQ) confirmed that INH stimulated autophagy but did not inhibit it by impairing lysosomal degradation. The blockage of autophagy with CQ exacerbated INH-induced apoptosis significantly. Further study showed that INH treatment down-regulated the protein phosphorylation of the mammalian target of rapamycin (mTOR), the key negative regulator of autophagy. In addition, INH induced p38 signaling activation. SB203580, a p38 inhibitor, effectively enhanced INH-induced apoptosis by increasing the cleavages of caspases 9, 3 and PARP, but did not affect autophagy. In summary, we firstly found that INH induced a protective autophagy which was associated with the inhibition of the mTOR pathway, and that INH induced p38 signaling activation to inhibit apoptosis by down-regulation of caspases 9, 3 and PARP pathways, but not that of autophagy. Thus, activation of autophagy and p38 signaling is presumably a therapeutic strategy for INH-induced hepatotoxicity.

[Mechanism of dracorhodin perchlorate-induced Hela cell apoptosis].

AIM: To study the mechanism of dracorhodin perchlorate-induced Hela cell apoptosis. METHODS: Cell viability was measured by MTT method. Morphological changes were observed by phase contrast microscopy and Hoechst 33258 staining. DNA fragmentation was assayed by agarose gel electrophoresis. Protein expression was detected by Western blot analysis. RESULTS: Dracorhodin perchlorate induced Hela cell apoptosis. The apoptosis was partially reversed by caspase-1, -3, -8, -9 and caspase family inhibitors. Treatment of Hela cells with dracorhodin perchlorate for 12 h increased the protein expression ratio of Bax/Bcl-XL; procaspase-3, -8, ICAD and PARP were cleaved to smaller molecules. CONCLUSION: Dracorhodin perchlorate induced Hela cell death via alteration of Bax/Bcl-XL ratio and activation of caspases.

Negatively-regulated necroptosis by autophagy required caspase-6 activation in TNFα-treated murine fibrosarcoma L929 cells.

Autophagy and necroptosis have been known to be interconnected, while the relationship between autophagy and necroptosis remains unclear. Here, we demonstrated that pan-caspase inhibitor z-VAD-fmk (zVAD) exacerbated TNFα-induced necroptosis and autophagy in murine fibrosarcoma L929 cells. And the RIP-1 inhibitor necrostatin-1 inhibited TNFα+zVAD-induced necroptosis and autophagy. Inhibition of autophagy by 3-methyladenine (3MA) or small interfering RNA (siRNA) against Beclin 1 augmented TNFα-induced necroptosis, while, autophagy inhibition did not influence TNFα+zVAD-induced necroptosis. These results suggested that autophagy was a downstream consequence of necroptosis, and had a negative-feedback function to necroptosis in TNFα-treated L929 cells, but not in the presence of zVAD. Subsequently, TNFα administration was accompanied with caspase-6 activation. Inhibition of caspase-6 activity by z-V-E(OMe)-I-D(OMe)-fmk (zVEID) or caspase-6 (p20) siRNA had no effect on necroptosis but promoted TNFα-induced autophagy. Meanwhile, autophagy inhibition further increased caspase-6 activation. Caspase-6 (p20) siRNA sequestered the increased necroptotic ratio by 3MA pretreatment in TNFα-treated L929 cells. In addition, caspase-6 activation induced by TNFα administration was inhibited by zVAD. Further, autophagy induced by higher concentration of zVAD did not negatively regulate necroptosis because caspase-6 was not activated. Collectively, our data indicated that autophagy was a downstream consequence of necroptosis, and negatively regulated necroptosis when caspase-6 was activated in TNFα-treated L929 cells.

Cytotoxic prenylated flavonoids from the stem bark of Maackia amurensis.

Five new prenylated flavonoids, maackiaflavanone A (1), maackiaflavanone B (2), maackiapentone (3), maackiapterocarpan A (4), maackiapterocarpan B (5) along with eleven known flavonoids were isolated from the stem bark of Maackia amurensis. The structures of the new compounds were elucidated by spectroscopic methods. The cytotoxicities of compounds 1-4, 6, 8-12 and 14-16 against four human cancer cell lines, A375S2, HeLa, MCF-7 and HepG2, were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Among the compounds tested, compound 2 showed the strongest cytotoxic activity with IC(50) value of 7.8 microM against A375S2 and euchrenone b(1) showed the most potent cytotoxicity with IC(50) value of 4.5 microM against HeLa.

Beta-eudesmol suppresses tumour growth through inhibition of tumour neovascularisation and tumour cell proliferation.

In the present study, we investigated the potential anti-angiogenic mechanism and anti-tumour activity of beta-eudesmol using in vitro and in vivo experimental models. Proliferation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor (VEGF, 30 ng/ml) and basic fibroblast growth factor (bFGF, 30 ng/ml) was significantly inhibited by beta-eudesmol (50-100 microM). Beta-eudesmol (100 microM) also blocked the phosphorylation of cAMP response element binding protein (CREB) induced by VEGF (30 ng/ml) in HUVEC. Beta-eudesmol (10-100 microM) inhibited proliferation of HeLa, SGC-7901, and BEL-7402 tumour cells in a time- and dose-dependent manner. Moreover, beta-eudesmol treatment (2.5-5 mg/kg) significantly inhibited growth of H(22) and S(180) mouse tumour in vivo. These results indicated that beta-eudesmol inhibited angiogenesis by suppressing CREB activation in growth factor signalling pathway. This is the first study to demonstrate that beta-eudesmol is an inhibitor of tumour growth.

Morphology of cultured human epidermal melanocytes observed by atomic force microscopy.

The objective of this study was to image the surface structure of cultured human epidermal melanocytes using atomic force microscopy (AFM). Epidermis obtained from human foreskins was treated with 0.5% dispase. Cell suspensions of the epidermis were prepared and seeded in six-well plates, in which sheets of mica had been placed. Samples for AFM were fixed on mica and scanning AFM images were captured by contacting and tapping modes operated under normal atmospheric pressure and temperature. Human epidermal melanocytes exhibited rounded, oval, triangular or quadrangular perikarya from which eight to 10 thick dendrites arose. These dendrites first bifurcated near the soma and then divided profusely into daughter branches, which spread out in all directions. We observed string-like long thin projections, growth cones and shorter thicker projections, which arose from the dendritic shafts, in which groups of melanosomes were arrayed. In addition to such structures, the most striking feature was the presence of filopodia arising from the melanocyte dendrite tips and the melanocyte cell body, many of which contained melanosomes. The termini of dendrites formed unbranched terminal protrusions (approximately 1,500-2,000 nm wide) consisting of two to three melanosomes wrapped in an arc, with their filopodia extending outwards. The tips of these structures also appeared to be squeezed and finally pinched off by the melanocyte to form a pouch filled with numerous melanosomes. We conclude that secondary and tertiary branches and subordinate branches might take part in transferring melanosomes into keratinocytes in addition to the transfer through the tips of the dendritic shafts. The melanin granules were expelled by exocytosis.