Ethanol Enhances Hyperthermia-Induced Cell Death in Human Leukemia Cells.

Ethanol has been shown to exhibit therapeutic properties as an ablative agent alone and in combination with thermal ablation. Ethanol may also increase sensitivity of cancer cells to certain physical and chemical antitumoral agents. The aim of our study was to assess the potential influence of nontoxic concentrations of ethanol on hyperthermia therapy, an antitumoral modality that is continuously growing and that can be combined with classical chemotherapy and radiotherapy to improve their efficiency. Human leukemia cells were included as a model in the study. The results indicated that ethanol augments the cytotoxicity of hyperthermia against U937 and HL60 cells. The therapeutic benefit of the hyperthermia/ethanol combination was associated with an increase in the percentage of apoptotic cells and activation of caspases-3, -8 and -9. Apoptosis triggered either by hyperthermia or hyperthermia/ethanol was almost completely abolished by a caspase-8 specific inhibitor, indicating that this caspase plays a main role in both conditions. The role of caspase-9 in hyperthermia treated cells acquired significance whether ethanol was present during hyperthermia since the alcohol enhanced Bid cleavage, translocation of Bax from cytosol to mitochondria, release of mitochondrial apoptogenic factors, and decreased of the levels of the anti-apoptotic factor myeloid cell leukemia-1 (Mcl-1). The enhancement effect of ethanol on hyperthermia-activated cell death was associated with a reduction in the expression of HSP70, a protein known to interfere in the activation of apoptosis at different stages. Collectively, our findings suggest that ethanol could be useful as an adjuvant in hyperthermia therapy for cancer.

Anticancer properties of the abietane diterpene 6,7-dehydroroyleanone obtained by optimized extraction.

AIM: 6,7-dehydroroyleanone (DHR) is a cytotoxic abietane present in the essential oil of Plectranthus madagascariensis. METHODS/RESULTS: Different extraction parameters were tested, and its extraction optimization was accomplished with a Clevenger apparatus-based hydrodistillation. After isolation, its effect on microtubules, P-glycoprotein and caspases was assessed on several cell lines and the compound was coupled with hybrid nanoparticles. The results show that DHR does not interfere with microtubule formation, but evades the resistance mechanisms of P-glycoprotein. Strong activation of caspases-3 and -9 indicates that DHR is able to induce apoptosis by triggering the intrinsic cell death pathway. Moreover, the assembly of DHR with hybrid nanoparticles was able to potentiate the effect of DHR in cancer cells. CONCLUSION: DHR seems to be a promising starting material with anticancer properties to further be explored.

Recent Advances on Cytotoxic Sesquiterpene Lactones.

Sesquiterpene lactones are naturally occurring compounds that have attracted considerable attention because to their vast array of biological activities. These plant-derived compounds contain the α-methylene-γ- butyrolactone functional group, which is the structural requirement for their pharmacological activities. Many of them exhibit cancer cell cytotoxicity and are promising anticancer agents through multiple mechanisms of action. Sesquiterpene lactones are alkylating agents that form covalent adducts in vivo and inhibit enzymes and key proteins. They are also potent apoptotic inducers in several cancer cells. This kind of cell death is recognized as a property that is useful for identifying anticancer drugs. This review focuses on the advances on cytotoxic sesquiterpene lactones and specifically the signal transduction pathways of cell death triggered in cancer cells.

The abietane diterpenoid parvifloron D from Plectranthus ecklonii is a potent apoptotic inducer in human leukemia cells.

BACKGROUND: Abietane diterpenes have attracted much attention because they display a wide range of biological activities, including antitumor activities. These compounds are the most diverse of the diterpenoids isolated from species of Plectranthus. Naturally occurring diterpene parvifloron D is the main phytochemical constituent of Plectranthus ecklonii. To examine the therapeutic potential of the plant, we evaluated whether parvifloron D displays cytotoxicity against human tumor cells. METHODS: The cytotoxicity was analyzed by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was evaluated by fluorescent microscopy, transmission electron microscopy, flow cytometric analysis of annexin V-FITC and propidium iodide-stained cells and DNA fragmentation. Protein expression and processing and release of mitochondrial proteins were analyzed by Western blot. Caspase activity was determined using colorimetric substrates. The membrane potential and intracellular reactive oxygen species were detected by flow cytometry. RESULTS: Parvifloron D displays strong cytotoxic properties against leukemia cells (HL-60, U-937, MOLT-3 and K-562) and in particular P-glycoprotein-overexpressing K-562/ADR cells, but has only weak cytotoxic effects on peripheral blood mononuclear cells (PBMCs). Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-xL did not confer resistance to parvifloron D-induced cytotoxicity. Growth inhibition of HL-60 cells that was triggered by parvifloron D was found to be caused by a rapid induction of apoptotic cell death. This apoptosis was prevented by the non-specific caspase inhibitor z-VAD-fmk, and by the selective caspase-9 inhibitor z-LEHD-fmk. Cell death induced by parvifloron D was found to be (i) associated with the dissipation of the mitochondrial membrane potential and the release of cytochrome c, (ii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 signaling and (iii) caused by a mechanism dependent on intracellular reactive oxygen species generation. CONCLUSION: Parvifloron D is a potent cytotoxic compound against several human tumor cells and also a fast and potent apoptotic inducer in leukemia cells.

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.

Melatonin induces apoptosis through a caspase-dependent but reactive oxygen species-independent mechanism in human leukemia Molt-3 cells.

Melatonin is a naturally occurring indoleamine synthesized in the pineal gland that exhibits an extensive repertoire of biological activities. An increasing number of studies indicate that melatonin protects normal cells, while it reducing cancer cell proliferation. In this study, we investigated the effect of melatonin on the growth of the human leukemia cells and found that it efficiently reduced the number of cells in a concentration- and time-dependent manner. Thus, incubation with the indoleamine increased the percentage of cells with a hypodiploid DNA content, augmented the number of annexin V-positive cells, and also provoked ultrastructural changes that are features of apoptotic cell death. Evaluation of caspases revealed that caspase-3, caspase-6, caspase-7, and caspase-9, but not caspase-8 and caspase-2, were quickly activated (3-6 hr). The increase in the activity of these proteases was associated with up-regulation of the pro-apoptotic factor Bax and also with the release of cytochrome c from mitochondria. Pretreatment of the cells with the general caspase inhibitor z-VAD-fmk, reduced melatonin-induced apoptosis, but it did not block cell death suggesting that melatonin activates an alternative cell death modality in the absence of caspase activity. Thus, the activation of caspases was preceded by a fast (<30 min) increase in reactive oxygen species (ROS). Rotenone and antimycin A reduced the levels of ROS stimulated by melatonin, indicating that the complex I and the complex III of the mitochondrial electron transport chain are important sources of these chemical species. However, the role of ROS in melatonin-induced cell death remains elusive because anti-oxidants that were shown to decrease ROS levels (glutathione, N-acetyl-l-cysteine and Trolox) were unable to abrogate melatonin-induced cell death.

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.

Pregnane steroidal glycosides and their cytostatic activities.

Four new steroidal glycosides such as 3-O-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside-12-ß-tigloyl-14-ß-hydroxy-17-ß-pregnane (1), 3-O-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside-12-ß-(2'-amino)-benzoyl-14-ß-hydroxy-17-ß-pregnane (2), 3-O-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside-12-ß-14-ß-dihydroxy-17-α-pregnane (3) and 3-O-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside-12-ß-14-ß-dihydroxy-17-ß-pregnane (4) were isolated from the aerial parts of Ceropegia fusca Bolle (Asclepiadaceae), a crassulacean acid metabolism plant, an endemic species to the Canary Islands that has been used in traditional medicine as a cicatrizant, vulnerary and disinfectant. The dichloromethane extract exhibited significant cytostatic activity against HL-60, A-431 and SK-MEL-1 cells, human leukemic, epidermoid carcinoma and melanoma cells, respectively. As shown in Table I, compounds 1 and 2 showed very similar IC(50) values. The acetylation of 1 to give the diacetate 5 increases 5-fold the cytotoxicity against HL-60 cells. Compounds 3 and 4 did not show cytotoxicity at the assayed concentrations. With respect to the compounds containing only the steroid ring (6-8), the presence of a charged O-amino-benzoyl but not a tigloyl group improved the cytotoxicity.

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.

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).

Cytotoxic activities of flavonoid glycoside acetates from Consolida oliveriana.

The flavonoids kaempferol, quercetin, trifolin, hyperoside 2''- and 6''- acetates, 7-glucotrifolin, biorobin and robinin were isolated from the aerial parts of Consolida oliveriana. Their derivatives kaempferol tetraacetate, quercetin pentaacetate, trifolin heptaacetate and hyperoside octaacetate exhibited significant cytotoxicity IN VITRO against three human cell lines HL-60, U937 and SK-MEL-1 while hyperoside 2''-acetate, hyperoside-6''-acetate, glucotrifolin decaacetate and heptamethyltrifolin were inactive.

A homo-isoflavonoid and a cytotoxic saponin from Dracaena draco.

Two new compounds, dracol (= (3R)-2,3-dihydro-3,5-dihydroxy-7-methoxy-3-[(4-methoxyphenyl)methyl]-8-methyl-4H-[1]benzopyran-4-one; 1) and icodeside (= (1beta,3beta,23S,24S)-3,23-dihydroxy-1-{[2-O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl)-alpha-L-arabinopyranosyl]oxy}spirosta-5,25(27)-dien-24-yl alpha-L-arabinopyranoside; 2), were isolated from the EtOH extract of the leaves of Dracaena draco, together with 17 known constituents. The structures of 1 and 2 were elucidated by in-depth spectroscopic analysis, and those of the known compounds were identified by comparison of their NMR and MS data with those reported in the literature. Icodeside (2) showed moderate cytotoxicity against human HL-60 and A-431 cells (Table 3).

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.

Childhood and malaria vaccines combined in biodegradable microspheres produce immunity with synergistic interactions.

Biodegradable microspheres may represent a potential tool for the delivery of combination vaccines. We demonstrate strong immunogenicity of five co-encapsulated antigens after a single subcutaneous inoculation in guinea pigs. Tetanus- and diphtheria-specific antibodies were not significantly affected by the presence of either antigen or by the presence of pertussis or Haemophilus influenzae type b (Hib) antigens. Microsphere formulations gave better protection against diphtheria toxin than did two injections of a licensed tetravalent vaccine. Finally, a synthetic malaria peptide antigen (PfCS) also encapsulated in PLGA microspheres increased diphtheria and tetanus-specific immunity and improved protection against diphtheria. These findings demonstrate the potential of microspheres as an alternative and promising strategy for combination vaccines with a further aptitude in reducing the number of inoculations required to gain functional immunity.

Steroidal saponins from the bark of Dracaena draco and their cytotoxic activities.

From the stem bark of Dracaena draco, three new compounds, namely, draconins A-C (1-3), were isolated, along with 17 known compounds. The structures of the new compounds isolated were elucidated on the basis of spectroscopic data interpretation. Several of the isolated compounds showed potent cytotoxic activities measured on the human leukemia cell line HL-60 (IC(50)'s from 2.0 to 9.7 microM at 72 h). The mechanism by which compounds 1 and 2 display their cytostatic properties is through induction of cell death by apoptosis, as evaluated by fluorescence microscopy and DNA fragmentation.