Sesquiterpenoids Isolated from Two Species of the Asteriscus Alliance.

Investigation of the aerial parts of two Spanish members of the Asteriscus alliance, Asteriscus graveolens subsp. stenophyllus and Asteriscus schultzii, afforded four new sesquiterpene lactones containing a humulene skeleton (1-4) and one new sesquiterpene lactone of the asteriscanolide type (5). Their chemical structures were determined on the basis of the HRMS and from 1D and 2D NMR spectroscopic studies. Both species showed different profiles of sesquiterpenoid constituents. A. schultzii did not show humulene or asteriscane sesquiterpenes, suggesting a resemblance to the genus Pallenis, another member of the Asteriscus alliance. A literature review on chemical isolates from the Asteriscus alliance supported the placement of A. schultzii in the genus Pallenis. The isolated components (1-5) were assessed for cytotoxicity against the HL-60 and MOLT-3 leukemia cell lines, with compound 1 showing activity in both biological assays (IC50 value range 4.1-5.4 µM).

ent-Labdane Diterpenoids from the Aerial Parts of Eupatorium obtusissmum.

Six new ent-labdane diterpenoids, uasdlabdanes A-F (1-6), were isolated from the aerial parts of Eupatorium obtusissmum. The new structures were elucidated through spectroscopic and spectrometric data analyses. The absolute configurations of compounds 1 and 2 were established by X-ray crystallography, and those of 3-6, by comparison of experimental and calculated electronic circular dichroism spectra. The antiproliferative activity of the compounds was studied in a panel of six representative human solid tumor cell lines and showed GI50 values ranging from 19 to >100 µM.

Ayanin diacetate-induced cell death is amplified by TRAIL in human leukemia cells.

Here we demonstrate that the semi-synthetic flavonoid ayanin diacetate induces cell death selectively in leukemia cells without affecting the proliferation of normal lymphocytes. Incubation of human leukemia cells with ayanin diacetate induced G(2)-M phase cell cycle arrest and apoptosis which was prevented by the non-specific caspase inhibitor z-VAD-fmk and reduced by the overexpression of Bcl-x(L). Ayanin diacetate-induced cell death was found to be associated with: (i) loss of inner mitochondrial membrane potential, (ii) the release of cytochrome c, (iii) the activation of multiple caspases, (iv) cleavage of poly(ADP-ribose) polymerase and (v) the up-regulation of death receptors for TRAIL, DR4 and DR5. Moreover, the combined treatment with ayanin diacetate and TRAIL amplified cell death, compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.

Secondary metabolites from two species of Tolpis and their biological activities.

Phytochemical research of two Tolpis species, T. proustii and T. lagopoda, led to the isolation of three new compounds: 30-chloro-3β-acetoxy-22α-hydroxyl-20(21)-taraxastene (1), 3β,22α-diacetoxy-30-ethoxy-20(21)-taraxastene (2) and 3β,28-dihydroxy-11α-hydroperoxy-12-ursene (3). The structures of the new compounds were elucidated by means of extensive IR, NMR, and MS data and by comparison of data reported in the literature. The in vitro antioxidant activities of the extracts were assessed by the DPPH and ABTS scavenging methods. The cytotoxicity of several known compounds and its derivatives was also assessed against human myeloid leukemia K-562 and K-562/ADR cell lines.

Secondary metabolites from two species of Pulicaria and their cytotoxic activity.

Two new compounds, the sesquiterpene (1E,5E)-8ß-acetoxy-4α-hydroxy-7ßH-germacra-1(10),5-dien-14-oic acid (2), and a nor-sesquiterpene, (5E)-8ß-acetoxy-4α-hydroxy-7ßH-germacr-5-en-10-one (3), were isolated from Pulicaria canariensis ssp. lanata, along with ten known compounds, including the flavonoid 5,3'-dihydroxy-3,7,4'-trimethoxyflavone (4). From Pulicaria burchardii, we isolated seven known compounds; the physical and spectroscopic data of the triterpenoid 3ß-hydroxytaraxaster-20-en-30-al (1) are reported. The structures of compounds 1-3 were determined on the basis of HR-MS, and 1D- and 2D-NMR studies. The structure of 2 was corroborated by X-ray crystal diffraction. Cell viability experiments revealed that the semisynthetic flavonoid 4b was the most cytotoxic compound against human leukemia cells, and the cytotoxicity was caused by induction of apoptosis, as determined by microscopy of nuclear changes.

Betuletol 3-methyl ether induces G(2)-M phase arrest and activates the sphingomyelin and MAPK pathways in human leukemia cells.

Betuletol 3-methyl ether (BME) is a natural phenylbenzo-gamma-pyrone that inhibits cell proliferation in human tumor cell lines and induces apoptotic cell death in HL-60 cells. Here we show that BME displays strong cytotoxic properties in several human leukemia cell lines (U937, K-562, THP-1, Jurkat, and Molt-3) and in cells that over-express two anti-apoptotic proteins, namely Bcl-2 and Bcl-x(L). BME arrested HL-60 cells at G(2)-M phase of the cell cycle, which was associated with the accumulation of cyclin B1 and p21(Cip1). Fluorescence microscopy experiments suggest that BME blocked the cell cycle in mitosis. The in vivo tubulin polymerization assay shows that BME inhibits tubulin polymerization and causes similar changes of cellular microtubule network as colchicine. Our results demonstrate that BME-induced cell death is (i) triggered in human myeloid leukemia cell that over-express Bcl-2 and Bcl-x(L), and (ii) associated with loss of inner mitochondrial membrane potential (DeltaPsim) and an increase in reactive oxygen species (ROS). Although ROS increased in response to BME, this did not seem to play a pivotal role in the apoptotic process since the anti-oxidant trolox was unable to provide cell protection. The treatment of HL-60 cells with BME induces the activation of mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinases, p38 mitogen-activated protein kinases and extracellular signal-regulated kinases (ERK)1/2 and stimulates the acid sphingomyelinase with concomitant ceramide generation. The findings of this study suggest that BME could be useful in the development of novel anticancer agents.

Naturally occurring asteriscunolide A induces apoptosis and activation of mitogen-activated protein kinase pathway in human tumor cell lines.

Sesquiterpene lactones have attracted much attention because they display a wide range of biological activities, including antitumor properties. Here, we show the effects of the naturally occurring sesquiterpene lactone asteriscunolide A (AS) on viability of human melanoma, leukemia and cells that overexpress antiapoptotic proteins, namely Bcl-2 and Bcl-x(L). All cell lines were sensitive to this compound, with IC(50) values of approximately 5 microM. The cytotoxic effects of AS were accompanied by a G(2)-M phase arrest of the cell cycle and a concentration- and time-dependent appearance of apoptosis as determined by DNA fragmentation, translocation of phosphatidylserine to the cell surface and sub-G(1) ratio. Apoptosis was associated with caspase-3 activity and poly(ADP-ribose) polymerase cleavage and was prevented by the nonspecific caspase inhibitor z-VAD-fmk, indicating that caspases are essential components in this pathway. The apoptotic effect of AS was also associated with (i) the release of cytochrome c from mitochondria which was accompanied by dissipation of the mitochondrial membrane potential (Delta Psi(m)) and (ii) the activation of the mitogen-activated protein kinases (MAPKs) pathway. AS-induced cell death was potentiated by inhibition of extracellular signal-regulated kinases (ERK) 1/2 signaling with U0126 and PD98059. Intracellular reactive oxygen species (ROS) seem to play a pivotal role in this process since high levels of ROS were produced early (1 h) and apoptosis was completely blocked by the free radical scavenger N-acetyl-L-cysteine (NAC). The present study demonstrates that AS-induced cell death is mediated by an intrinsic-dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism dependent on ROS generation.

Sesquiterpene lactones from Gonospermum gomerae and G. fruticosum and their cytotoxic activities.

Four new sesquiterpene lactones (1-4) and a new sesquiterpene (5) together with 20 known compounds were isolated from two Gonospermum species (G. gomerae Bolle and G. fruticosum Less). Their structures were determined by analysis of spectroscopic data, including 1D and 2D NMR. The cytotoxicity of several new and known natural and semisynthetic sesquiterpene lactones was also assessed against human myeloid leukemia cell lines (HL-60 and U937), human melanoma cells (SK-MEL-1), and human adenocarcinoma (A549).

Acetyl derivative of quercetin 3-methyl ether-induced cell death in human leukemia cells is amplified by the inhibition of ERK.

Flavonoids are polyphenolic compounds that are ubiquitously in plants and display a vast array of biological activities. Here we have studied the effect of the phenylbenzo-gamma-pyrone-derivative quercetin 3-methyl ether tetracetate (QD), obtained by acetylation of the natural product quercetin 3-methyl ether, on cell viability of human leukemia HL-60 and U937 cell lines. The results show that QD was cytotoxic and induced G2-M phase cell cycle arrest on both cell lines and it was a potent apoptotic inducer on HL-60 cells. QD-induced apoptosis is (i) mediated by caspase activation, since it was prevented by the non-specific caspase inhibitor z-VAD-fmk, (ii) associated with cytochrome c release and (iii) triggered in Bcl-2 over-expressing U937 cells. The treatment of HL-60 and U937 cells with QD also induces the activation of the mitogen-activated protein kinases (MAPKs) pathway, including c-Jun N-terminal kinase, p38 mitogen-activated protein kinase and extracellular signal-regulated kinases (ERK) 1/2. Inhibition of c-Jun N-terminal kinase by SP600125 and of p38 mitogen-activated protein kinase by SB203580 had no influence on QD-mediated apoptosis. In contrast, inhibition of ERK1/2 with the pharmacologic inhibitors U0126 or PD98059, together with QD, resulted in an important enhancement of apoptosis. Cells are sensitized to QD-mediated apoptosis after blocking ERK1/2, which suggests that inhibition of this pathway is a valuable strategy to increase the sensitivity of human leukemia HL-60 cells toward QD.

Phenylbenzopyrones structure-activity studies identify betuletol derivatives as potential antitumoral agents.

We have analyzed the cytotoxicity of 22 compounds with a phenylbenzo-gamma-pirone core structure, most of them obtained from natural sources, in five human tumor cell lines (HL-60, A431, SK-OV-3, HeLa and HOS). Betuletol 3-methyl ether and its diacetate were the most cytotoxic compounds. The HL-60 cell line was especially sensitive to these compounds, with IC50 values of approximately 1 microM. Treatment of HL-60 cells with betuletol 3-methyl ether was associated with apoptosis induction which was prevented by a non-specific caspase inhibitor (z-VAD-fmk) and also by a specific inhibitor of caspase-8 (z-IETD-fmk) indicating activation of the extrinsic apoptotic pathway. The results suggest that betuletol 3-methyl ether has potential as new anticancer agent.

The eudesmanolide tanapsin from Tanacetum oshanahanii and its acetate induce cell death in human tumor cells through a mechanism dependent on reactive oxygen species.

In this study the cytotoxicities of two species of Tanacetum were evaluated against human tumor cells. Tanacetum oshanahanii extract was more cytotoxic than Tanacetum ptarmiciflorum. Analyses of both extracts of Tanacetum by ultrahigh performance liquid chromatography-tandem mass spectrometry revealed that T. oshanahanii extract contains the eudesmanolide tanapsin, while T. ptarmiciflorum lacks this sesquiterpene lactone. Tanapsin was cytotoxic against leukemia and melanoma cells, including cells that overexpress Bcl-2 and Bcl-xL, with IC50 values of approximately 10 µM, but not against quiescent or proliferating human peripheral blood mononuclear cells. Treatment of cells with tanapsin induced apoptosis. This was prevented by the non-specific caspase inhibitor z-VAD-fmk, and reduced by the selective caspase-3/7 inhibitor z-DEVD-fmk. Tanapsin acetate was also cytotoxic against leukemia and melanoma cells and a potent apoptotic inducer. Tanapsin-induced cell death was found to be associated with (i) the loss of inner mitochondrial membrane potential (ΔΨm) and release of mitochondrial cytochrome c, (ii) the activation of multiple caspases and the mitogen-activated protein kinase pathway, and (iii) an increase in reactive oxygen species generation. Generation of reactive oxygen species in response to tanapsin seems to play a crucial role in the cell death process since the antioxidant N-acetyl-l-cysteine blocked both ROS generation and cell death.

Potent induction of apoptosis by germacranolide sesquiterpene lactones on human myeloid leukemia cells.

This paper studies the cytotoxic effect induced by four known natural sesquiterpene lactones (tatridin A, tamirin, reynosin, ineupatorolide A) and one synthetic derivative (tatridin A diacetate) on the myeloid leukemia cell lines HL-60 and U937. Tatridin A diacetate and ineupatorolide A were found to be the most cytotoxic compounds with growth inhibition caused by induction of apoptosis as determined by flow cytometry and microscopy of nuclear changes. The results reported here support the conclusion that apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase-1 and was also associated with an early release of cytochrome c from the mitochondria.

A chemotaxonomic study of endemic species of genus Tanacetum from the Canary Islands.

Aerial parts of Tanacetum oshanahanii collected at "Jardín Canario Viera y Clavijo", Tanacetum ptarmiciflorum collected at Los Moriscos (Tejeda), and Tanacetum ferulaceum var. latipinnum collected at Anden Verde (Agaete) in Gran Canaria, Canary Islands, afforded three sesquiterpenes related to nerolidol and six sesquiterpene lactones whose structures were established on the basis of their spectroscopic data and chemical transformations. In this work we show that this type of sesquiterpene lactones could be used as chemotaxonomic markers. A series of sesquiterpene lactones described in this paper were assessed for cytotoxicity against HL-60 and U937 cancer cell lines. The derivatives 106a and 98a displayed cytotoxic properties showing IC50 values between 5 and 11 µM. Furthermore, we demonstrated that these selected sesquiterpene lactones induce apoptotic cell death in human leukemia cells through a mechanism that involves activation of multiple caspases and moreover cell death was found to be associated with the release of cytochrome c.

Chemotaxonomy of Gonospermum and related genera.

Aerial parts of Gonospermum fruticosum collected at several locations in the Canary Islands afforded, in addition to known compounds, four sesquiterpene alcohols related to costol and a sesquiterpene lactone, whose structures were established on the basis of their spectroscopic data and chemical transformations. Except for Gonospermum species collected on the island of Tenerife, those collected on the island of El Hierro and, in a previous study those from La Gomera, contain sesquiterpene lactones that can be used as chemotaxonomic markers confirming the inclusion of Gonospermum, Lugoa, and species of Tanacetum endemic to the Canary Islands in a genus that does not support the monophyly of Gonosperminae.

Chemical constituents of Tolpis species.

Phytochemical research of two Tolpis species, T. webbii and T. sp., led to the isolation of three new compounds: 2,4'-dihydroxy-4-methoxybenzophenone (1) and the triterpenes 21 alpha, 22 alpha-epoxy-20 alpha-hydroxy-20(30)-dihydrotaraxasterol (2) and 3beta-hydroxytaraxaster-20-en-30-oic acid (3) together with 16 known compounds. The structures of the new compounds were elucidated by means of extensive IR, NMR, MS and X-ray analysis and by comparison of data reported in the literature.

Sesquiterpenoids from Pulicaria canariensis and their cytotoxic activities.

Thirteen new sesquiterpenes, pulicanadiene A (1), B (2), and C (3), pulicanone (4), pulicanol (5), pulicanarals A (6), B (7), and C (8), pulicanadienals A (9) and B (10), pulicanadienol (11), and pulioplopanones A (12) and B (13), and seven known compounds, stigmasterol, ergosterol peroxide, calenduladiol, 7,4'-di-O-methyldihydrokaempferol, 5,7-dihydroxy-3,3',4'-trimethoxyflavone, dihydroquercetin 7,3'-dimethyl ether, and 6,15alpha-epoxy-1beta,4beta-dihydroxyeudesmane, were isolated from Pulicaria canariensis. Compound 4a showed cytotoxicity on the human myeloid leukemia cell line HL-60. The cytotoxicity was caused by induction of apoptosis as determined by microscopy of nuclear changes, activation of caspases, and the cleavage of poly(ADP-ribose) polymerase-1.

Sesquiterpenoid derivatives from Gonospermum elegans and their cytotoxic activity for HL-60 human promyelocytic cells.

Four new compounds, a sesquiterpene, eleganodiol (1), and three sesquiterpene lactones, eleganolactone A (2), eleganolactone B (3), and elegain (4), were isolated from Gonospermum elegans along with 16 known compounds. The structures of 1, 2, and 4 were determined on the basis of MS and NMR studies of their acetate derivatives (1a, 2a, 4a). The structure of the acetate derivative (3a) of 3 was determined on the basis of spectroscopic data interpretation and by single-crystal X-ray diffraction. Compounds 2a and 3a were used to study their biological activities on the HL-60 human promyelocytic leukemia cell line. These compounds induced morphological changes and internucleosomal DNA fragmentation characteristic of apoptotic cell death.