Metal complexes of natural melophlins and their cytotoxic and antibiotic activities

Complexes of the natural melophlins A and C with Mg, Zn, Ga, La and Ru were prepared, characterized and tested for antimicrobial and cytotoxic effects. The lanthanum complex La(melophlinato C)₃ and the ruthenium complex chlorido(η⁶-p-cymene)(melophlinato C)ruthenium(II) inhibited cells of human A-498 kidney cancer at IC₅₀ = 0.54 μM and 1.0 μM, respectively, and so distinctly better than free melophlin C. Another synergistic effect of coordinating melophlins to bioactive metals was found in the growth inhibition of the melophlin C-resistant bacterium Micrococcus luteus by Ga, La and Ru complexes of melophlin C.     

Melanogenesis‐Inhibitory Saccharide Fatty Acid Esters and Other Constituents of the Fruits of Morinda citrifolia (Noni)

Five new saccharide fatty acid esters, named nonioside P ( 3 ), nonioside Q ( 4 ), nonioside R ( 8 ), nonioside S ( 10 ), and nonioside T ( 14 ), and one new succinic acid ester, butyl 2‐hydroxysuccinate (=4‐butoxy‐3‐hydroxy‐4‐oxobutanoic acid) ( 31 ), were isolated, along with 26 known compounds, including eight saccharide fatty acid esters, 1, 2, 5, 6, 7, 9, 12 , and 13 , three hemiterpene glycosides, 15, 17 , and 18 , six iridoid glycosides, 21 – 25 , and 27 , and nine other compounds, 20, 28, 29 , and 32 – 37 , from a MeOH extract of the fruit of Morinda citrifolia (noni). Upon evaluation of these and five other glycosidic compounds, 11, 16, 19, 26 , and 30 , from M. citrifolia fruit extract for their inhibitory activities against melanogenesis in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), most of the saccharide fatty acid esters, hemiterpene glycosides, and iridoid glycosides showed inhibitory effects with no or almost no toxicity to the cells. These compounds were further evaluated with respect to their cytotoxic activities against two human cancer cell lines (HL‐60 and AZ521) and their inhibitory effects on Epstein? Barr virus early antigen (EBV‐EA) activation induced with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells.     

Netamines O–S,Five New Tricyclic Guanidine Alkaloids from the Madagascar Sponge Biemna laboutei,and Their Antimalarial Activities

In our continuing program to isolate new compounds from the Madagascar sponge Biemna laboutei, five new tricyclic guanidine alkaloids, netamines O – S ( 1 – 5 , resp.), have been identified together with the known compounds netamine E ( 6 ) and mirabilin J ( 7 ). The structures of all new netamines were assigned on the basis of spectroscopic analyses. Their relative configurations were established by analysis of ROESY data and comparison with literature data. Netamines O, P, and Q, which were isolated in sufficient quantities, were tested for their cytotoxic activities against KB cells and their activities against the malaria parasite Plasmodium falciparum. Netamines O and Q were found to be moderately cytotoxic. Netamines O, P, and Q exhibited antiplasmodial activities with IC₅₀ values of 16.99±4.12, 32.62±3.44, and 8.37±1.35 μM , respectively.     

Pierisformotoxins A – D,Polyesterified Grayanane Diterpenoids from Pieris formosa and Their cAMP‐Decreasing Activities

Four highly acylated diterpenoids, designated as pierisformotoxins A–D ( 1 – 4 , resp.), along with 26 known compounds, were isolated from the flowers of Pieris formosa. Among them, pierisformotoxins A and B ( 1 and 2 , resp.) were new highly acylated grayanane diterpenoids, of which the five‐membered ring A has undergone an oxidative cleavage between C(3) and C(4), followed by lactonization, to give rise to a five‐membered lactone ring between C(3) and C(5), differing from the previously reported grayanane diterpenoids with a 5/7/6/5 ring system. Results of the cAMP‐regulation‐activity assay showed that pierisformotoxin C ( 3 ) at 10 μM (inhibitory ratio (IR): 10.1%) or 2 μM (9.8%), and pierisformotoxin B ( 2 ) at 50 μM (13.9%) significantly decreased the cAMP level in N1E‐115 neuroblastoma cells (p<0.05).     

Novel Zierane‐ and Guaiane‐Type Sesquiterpenes from the Root of Melicope denhamii

Two novel zierane‐type sesquiterpenes, named melicodenones A and B ( 1 and 2 , resp.), and three new guaiane‐type sesquiterpenes, named melicodenones C–E ( 3 – 5 ), were isolated from the root of Melicope denhamii (Seem. ) T. G. Hartley together with zierone ( 6 ). Their structures were established by extensive NMR‐spectroscopic analyses. Compounds 1 – 6 were tested for cytotoxicity using human colon cancer DLD‐1 cells, and melicodenone A ( 1 ) was found to exhibit moderate activity.     

Ecosystem development in short‐term postglacial chronosequences: N and P limitation in glacier forelands from Santa Inés Island,Magellan Strait

Glacier foreland moraines provide an ideal model to examine the patterns of ecosystem development and the evolution of nitrogen and phosphorous limitation over successional time. In this paper, we focus on a 400‐year soil chronosequence in the glacier forelands of Santa Inés Island in the Magellan Strait, southern Chile by examining forest development on phosphorus (P)‐poor substrates in a uniquely unpolluted region of the world. Results show a steady increase in tree basal area and a humped trend in tree species richness over four centuries of stand development. The increase in basal area suggests that the late successional tree species were more efficient nutrient users than earlier successional ones. Total contents of carbon (C) and nitrogen (N) in soils increased during the chronosequence, reaching an asymptote in late succession. The net increases in soil C : N, C : P and N : P ratios observed over successional time suggest that nutrient limitation is maximal in 400‐year‐old substrates. Foliar C : N and C : P ratios also increased over time to reach an asymptote in old‐growth stages, following soil stoichiometric relationships; however the foliar N‐to‐P ratio remained constant throughout the chronosequence. Biological N fixation was greater in early postglacial succession, associated with the presence of the symbiotic N‐fixer Gunnera magellanica. Declining trends of δ¹⁵N in surface soils through the 400‐year chronosequence are evidence of decreasing N losses in old‐growth forests. In synthesis, glacier foreland chronosequences at this high South American latitude provide evidence for increasing efficiency of N and P use in the ecosystem, with the replacement of shade‐intolerant pioneers by more efficient, shade‐tolerant tree species. This pattern of ecosystem development produces a constant foliar N : P ratio, regardless of variation in soil N‐to‐P ratio over four centuries.     

Disulfiram combined with copper inhibits metastasis and epithelial–mesenchymal transition in hepatocellular carcinoma through the NF‐κB and TGF‐β pathways

Late‐stage hepatocellular carcinoma (HCC) usually has a low survival rate because of the high risk of metastases and the lack of an effective cure. Disulfiram (DSF) has copper (Cu)‐dependent anticancer properties in vitro and in vivo. The present work aims to explore the anti‐metastasis effects and molecular mechanisms of DSF/Cu on HCC cells both in vitro and in vivo. The results showed that DSF inhibited the proliferation, migration and invasion of HCC cells. Cu improved the anti‐metastatic activity of DSF, while Cu alone had no effect. Furthermore, DSF/Cu inhibited both NF‐κB and TGF‐β signalling, including the nuclear translocation of NF‐κB subunits and the expression of Smad4, leading to down‐regulation of Snail and Slug, which contributed to phenotype epithelial–mesenchymal transition (EMT). Finally, DSF/Cu inhibited the lung metastasis of Hep3B cells not only in a subcutaneous tumour model but also in an orthotopic liver metastasis assay. These results indicated that DSF/Cu suppressed the metastasis and EMT of hepatic carcinoma through NF‐κB and TGF‐β signalling. Our study indicates the potential of DSF/Cu for therapeutic use.     

Expression and characterization of Flavikunin: A Kunitz‐type serine protease inhibitor identified in the venom gland cDNA library of Bungarus flaviceps

Trancriptomic analysis of the venom gland cDNA library of Bungarus flaviceps revealed Kunitz‐type serine protease inhibitor as one of the major venom protein families with three groups A, B, C. One of the group B isoforms named Flavikunin, which lacked an extra cysteine residue involved in disulfide bond formation in β‐bungarotoxin, was synthesized, cloned, and overexpressed in Escherichia coli. To decipher the structure‐function relationship, the P1 residue of Flavikunin, histidine, was mutated to alanine and arginine. Purified wild‐type and mutant Flavikunins were screened against serine proteases‐thrombin, factor Xa, trypsin, chymotrypsin, plasmin, and elastase. The wild‐type and mutant Flavikunin (H?R) inhibited plasmin with an IC ₅₀ of 0.48 and 0.35 µM, respectively. The in‐silico study showed that P1 residue of wild‐type and mutant (H?R) Flavikunin interacted with S1′ and S1 site of plasmin, respectively. Thus, histidine at the P1 position was found to be involved in plasmin inhibition with mild anticoagulant activity.     

Induction of apoptosis by withaferin A in human leukemia U937 cells through down-regulation of Akt phosphorylation

Withaferin A, a major chemical constituent of Withania somnifera, has been reported for its tumor cell growth inhibitory activity, antitumor effects, and impairing metastasis and angiogenesis. The mechanism by which withaferin A initiates apoptosis remains poorly understood. In the present report, we investigated the effect of withaferin A on the apoptotic pathway in U937 human promonocytic cells. We show that withaferin A induces apoptosis in association with the activation of caspase-3. JNK and Akt signal pathways play crucial roles in withaferin A-induced apoptosis in U937 cells. Furthermore, we have shown that overexpression of Bcl-2 and active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase-3, and PLC-γ1 cleavage by withaferin A. Taken together, our results indicated that the JNK and Akt pathways and inhibition of NF-κB activity were key regulators of apoptosis in response to withaferin A in human leukemia U937 cells.     

Signaling Defect in the Activation of Caspase-3 and PKCδ in Human TUR Leukemia Cells Is Associated with Resistance to Apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-β- -arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G₀/G₁of the cell cycle and an accumulation of a population in the sub-G₁phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measuredin vitroby enhanced metabolization of a fluorescence substrate andin vivoby cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cδ. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

Three New Phenylpropanoids from the Roots of Piper taiwanense and Their Inhibitory Activities on Platelet Aggregation and Mycobacterium tuberculosis

Bioassay‐guided fractionation of the active AcOEt‐soluble fraction from the roots of Piper taiwanense has led to the isolation of two new phenylpropanoids, taiwanensols A and B ( 1 and 2 , resp.), a new natural product, taiwanensol C ( 3 ), and 3‐acetoxy‐4‐hydroxy‐1‐allylbenzene ( 4 ). The compounds were obtained as two isomer mixtures ( 1 / 2 and 3 / 4 , resp.). Their structures were elucidated by spectroscopic analyses, including 1D‐ and 2D‐NMR spectroscopy and mass spectrometry, and by the comparison of their NMR data with those of related compounds. Compounds 1 – 4 were evaluated for their antiplatelet and antitubercular activities. The mixtures 1 / 2 and 3 / 4 showed potent inhibitory activities against platelet aggregation induced by collagen, with IC₅₀ values of 35.2 and 8.8 μM , respectively. In addition, 1 / 2 and 3 / 4 showed antitubercular activities against Mycobacterium tuberculosis H37Rv, with MIC values of 30.0 and 48.0 μg/ml, respectively.     

A New Cytotoxic Carbazole Alkaloid and Two New Other Alkaloids from Clausena excavata

One new carbazole alkaloid, excavatine A ( 1 ), and two additional new alkaloids, excavatine B ( 2 ) and excavatine C ( 3 ), were isolated from the stems and leaves of Clausena excavata Burm .f. (Rutaceae). Their structures were determined on the basis of detailed spectroscopic analyses, especially 2D‐NMR and HR‐EI‐MS data. Compounds 1 – 3 were tested for their cytotoxic activities against A549, HeLa, and BGC‐823 cancer cell lines, and for their antimicrobial activities against Candida albicans and Staphylococcus aureus. Only 1 exhibited cytotoxicity against A549 and HeLa cell lines with the IC₅₀ values of 5.25 and 1.91 μg/ml, respectively.     

Variation of Epimedins A – C and Icariin in Ten Representative Populations of Epimedium brevicornu Maxim., and Implications for Utilization

The concentration variations of main flavonoids, epimedins A–C and icariin, among ten representative populations of Epimedium brevicornu Maxim . were assessed by HPLC. The populations were collected during the flowering stage and included 419 individual samples. Remarkable variations within and among populations were detected. SXXA Population (see Fig. 1) was an outlier due to its significant low concentrations (<1.00–4.46 mg/g). But even without SXXA, significant concentration differences among populations were still observed in epimedin A (2.31–8.42 mg/g), epimedin B (6.67–55.7 mg/g), epimedin C (5.39–23.0 mg/g), icariin (8.50–39.9 mg/g), and their total (29.1–123 mg/g). All populations except SXXA showed much higher concentrations than the recommended standards (i.e. 5 mg/g for icariin and 13 mg/g for the total). A high‐concentration‐population structure, estimated both by principal component analysis (PCA) and unweighted pair group method with averaging (UPGMA) cluster analysis, based on Euclidean distances, was observed. Both methods allowed separation of the populations in four groups defined by the concentrations of four main flavonoids. The populations (SXLC and SXQS) located in north of Yellow River were clustered together and characterized by highest concentrations of epimedin B, icariin, and their total. Considering of the high concentrations of main flavonoids and abundant resources, E. brevicornu could be exploited as a good medical resource for Herba Epimedii and would offer a tremendous potential for commercial development, but SXXA population should be paid special attention, and further study is needed.     

Kaempferol induces apoptosis in two different cell lines via Akt inactivation,Bax and SIRT3 activation,and mitochondrial dysfunction

Kaempferol (3,4′,5,7‐tetrahydroxyflavone) is a flavonoid with anti‐ and pro‐oxidant activity present in various natural sources. Kaempferol has been shown to posses anticancer properties through the induction of the apoptotic program. Here we report that treatment of the chronic myelogenous leukemia cell line K562 and promyelocitic human leukemia U937 with 50 µM kaempferol resulted in an increase of the antioxidant enzymes Mn and Cu/Zn superoxide dismutase (SOD). Kaempferol treatment induced apoptosis by decreasing the expression of Bcl‐2 and increasing the expressions of Bax. There were also induction of mitochondrial release of cytochrome c into cytosol and significant activation of caspase‐3, and ‐9 with PARP cleavage. Kaempferol treatment increased the expression and the mitochondria localization of the NAD‐dependent deacetylase SIRT3. K562 cells stably overexpressing SIRT3 were more sensitive to kaempferol, whereas SIRT3 silencing did not increase the resistance of K562 cells to kaempferol. Inhibition of PI3K and de‐phosphorylation of Akt at Ser473 and Thr308 was also observed after treating both K562 and U937 cells with kaempferol. In conclusion our study shows that the oxidative stress induced by kaempferol in K562 and U937 cell lines causes the inactivation of Akt and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and SIRT3, decrease of Bcl‐2, release of cytochrome c, caspase‐3 activation, and cell death. J. Cell. Biochem. 106: 643–650, 2009. © 2009 Wiley‐Liss, Inc.     

Triterpene Glucosides from the Leaves of Aralia elata and Their Cytotoxic Activities

Three new triterpene glucosides, named congmuyenosides C–E ( 1 – 3 , resp.), along with four known ones, were isolated from an EtOH extract of Aralia elata (Miq .) Seem . leaves. The structures of the new compounds were identified as 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}caulophyllogenin ( 1 ), 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}hederagenin 28‐O‐β‐D ‐glucopyranosyl ester ( 2 ), 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}echinocystic acid 28‐O‐β‐D ‐glucopyranosyl ester ( 3 ) on the basis of spectral analyses, including MS, ¹H‐NMR, ¹³C‐NMR, DEPT, HSQC, HMBC, NOESY, and HSQC‐TOCSY experiments. All isolates obtained were evaluated for their cytotoxic activities against three human tumor cell lines (HepG2, SKOV3, and A549). Compound 3 showed significant cytotoxicity against A549 cell line (IC₅₀ 9.9±1.5 μM ).     

Triterpenoids from the Stem Bark of Melia toosendan and Determination of Their Absolute Configurations at C(24)

Six new triterpenoids, meliasenins S–X ( 1 – 6 , resp.), were isolated from the stem bark of Melia toosendan. Their structures were elucidated by mass spectrometry, NMR experiments, and comparison with the known compounds. Particularly, the absolute configuration at C(24) in new compounds was determined through their CD spectra of the [Pr(FOD)₃] complex (fod=1,1,1,2,2,3,3,7,7,7‐decafluoroheptane‐4,6‐dione) in CCl₄, as well as by using Mosher's method.     

Growth and phosphate uptake kinetics of the cyanobacterium,Cylindrospermopsis raciborskii (Cyanophyceae) in throughflow cultures

• 1 Cylindrospermopsis raciborskii occupies a rapidly expanding geographical area. Its invasive success challenges eutrophication control in many lakes. To understand better the load‐dependent behaviour of this nitrogen fixing cyanobacterium under in situ conditions, we studied P‐dependent growth of a C. raciborskii strain under continuous and pulsed P supply.
• 2 The Droop model reasonably described P‐dependent growth in the continuously supplied chemostats. Large P pulses, however, caused a delay in growth and cells subject to P pulses grew slower than their counterparts with the same P quota supplied continuously.
• 3 The kinetics of P uptake indicated that C. raciborskii is opportunistic with respect to P. Its high excess P storage capacity after a saturating P pulse (Q_ex=95 µg P [mg C]^‐1) and P‐specific uptake capacity (U_max = V_max/Q_P=150–1200) are indicative of storage adaptation. At the same time, the affinity of the P uptake system (U_max/K = 800–4000) is also high.
• 4 Rate of leakage exceeded that of the steady state net P uptake by one to two orders of magnitude. Growth affinity of C. raciborskii (µ_max/K_µ≈ 20) was relatively low, presumably due to the substantial leakage.
• 5 The dynamics of the particular water body determine which trait contributes most to competitive success of C. raciborskii. In deep lakes with vertical nutrient gradients, the cyanobacterium may rely primarily on its high P storage capacity, which is coupled to a lack of short‐term feedback inhibition and efficient buoyancy regulation. In lakes without such gradients, high P uptake affinity may be vitally important.

     

SIRT3, PP2A and TTP protein stability in the presence of TNF‐α on vincristine‐induced apoptosis of leukaemia cells

The contribution of vincristine (VCR)‐induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL‐60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up‐regulation of TNF‐α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down‐regulated SIRT3, and such down‐regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1‐modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3‐ROS‐p38 MAPK‐PP2A axis inhibited tristetraprolin (TTP)‐controlled TNF‐α mRNA degradation, consequently, up‐regulating TNF‐α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS‐p38 MAPK axis increased the survival of VCR‐treated cells and repressed TNF‐α up‐regulation. In contrast to suppression of the ROS‐p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL‐60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3‐ROS‐p38 MAPK‐PP2A‐TTP axis modulates TNF‐α expression, which triggers apoptosis of VCR‐treated U937 and HL‐60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR‐elicited microtubule destabilization.     

Tirucallane Triterpenoids from the Stems of Brucea mollis

Three new tirucallane triterpenoids, brumollisols A–C ( 1 – 3 , resp.), together with five known analogues, (23R,24S)‐23,24,25‐trihydroxytirucall‐7‐ene‐3,6‐dione ( 4 ), piscidinol A ( 5 ), 24‐epipiscidinol A ( 6 ), 21α‐methylmelianodiol ( 7 ), and 21β‐methylmelianodiol ( 8 ), were isolated from an EtOH extract of the stems of Brucea mollis. Their structures were elucidated by means of spectroscopic methods including 1D‐ and 2D‐NMR techniques and mass spectrometry. In the in vitro assays, compound 6 exhibited significant cytotoxic activity against A549 and BGC‐823 cancer cells with IC₅₀ values of 1.16 and 3.01 μM , respectively. At a concentration of 10 μM , compounds 1 – 5, 7 , and 8 were found to inhibit NO production in mouse peritoneal macrophages with inhibitory ratios ranging from 39.8±7.7 to 68.2±4.5%.     

Determination of the Absolute Configuration of Two Pairs of C‐8 − C‐9′ Linked Neolignan Enantiomers

Two pairs of new neolignan enantiomers, (±)‐torreyayunan A ( 1a / 1b ) and (±)‐torreyayunan B ( 2a / 2b ), featuring a rare C‐8 ? C‐9′ linked skeleton, were isolated from leaves and twigs of Torreya yunnanensis. Their absolute configuration involving two chiral centers was determined by combined spectral and Density Functional Theory (DFT) calculation. This is the first report of the absolute configuration of this group of neolignans. Chirality 26:825–828, 2014. © 2014 Wiley Periodicals, Inc.