Secondary metabolites from Isodon ternifolius (D. Don) Kudo and their anticancer activity as DNA topoisomerase IB and Tyrosyl-DNA phosphodiesterase 1 inhibitors.

Based on DNA topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibition of the ethanol extract of the roots of Isodon ternifolius (D. Don) Kudo (Labiatae), its secondary metabolites has been studied. Two new compounds, an ent-abietane diterpenoid isodopene A (1) and a 2,3-seco-triterpene isodopene B (13), along with 25 known compounds were isolated. Their structures were elucidated by spectroscopic analysis and theoretical calculations. The enzyme-based assays indicated that 1 and 13 showed strong (+++) and moderate (++) TOP1 inhibition, respectively. Two chalcone derivatives 11 and 12 were firstly found as dual TDP1 and TOP1 natural inhibitors, and showed synergistic effect with the clinical TOP1 inhibitors topotecan in MCF-7 cells. Compounds 8, 16, and 22 acted as TOP1 catalytic inhibitors with equipotent TOP1 inhibition to camptothecin (++++). Compounds 7 and 8 exhibited significant cytotoxicity against MCF-7, A549, and HCT116 cells with GI50 values in the range of 2.2-4.8 µM. This work would provide valuable information that secondary metabolites from I. ternifolius could be developed as anticancer agents.

Lamellarin alkaloids: Isolation, synthesis, and biological activity.

Lamellarins are marine alkaloids containing fused 14-phenyl-6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinoline or non-fused 3,4-diarylpyrrole-2-carboxylate ring systems. To date, more than 50 lamellarins have been isolated from a variety of marine organisms, such as mollusks, tunicates, and sponges. Many of them, especially fused type I lamellarins, exhibit impressive biological activity, such as potent cytotoxicity, topoisomerase I inhibition, protein kinases inhibition, and anti-HIV-1 activity. Due to their useful biological activity and limited availability from natural sources, a number of synthetic methods have been developed. In this chapter, we present an updated and comprehensive review on lamellarin alkaloids summarizing their isolation, synthesis, and biological activity.

Repurposing of ginseng extract as topoisomerase I inhibitor based on the comparative analysis of gene expression patterns.

Repositioning of plant extracts and chemical drugs can accelerate drug development. However, its success rate may depend on what the clue is for the repositioning. Recently, repositioning based on correction of unwarranted gene expression pattern has suggested the possibility of new drug development. Here, we designed a similar method for the repositioning of nutraceutical ginseng (Panax ginseng C.A.Mey.), which is one of the most validated natural therapeutic products for various diseases. We analyzed ginseng-induced gene expression profiles using the connectivity map algorithm, which is a database that connects diseases, chemical drugs, and gene expression. Ginseng was predicted to show the same effects as those of topoisomerase I inhibitors. In a subsequent in vitro assay, ginseng extract unwound coiled or supercoiled DNA, an effect comparable to that of the topoisomerase I inhibitor, camptothecin. Furthermore, ginseng extract induced synthetic lethality with suppression of the Werner syndrome gene. The collected data implicate ginseng as a candidate antitumor agent owing to its topoisomerase I inhibitory activity and further validate the usefulness of differentially expressed gene similarity-based repurposing of other natural products.

Molecular Networking Reveals Two Distinct Chemotypes in Pyrroloiminoquinone-Producing Tsitsikamma favus Sponges.

The temperate marine sponge, Tsitsikamma favus, produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of T. favus sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as T. favus and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo-ortho-quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a Tsitsikamma sponge species, and the first description of distinct chemotypes within a species of the Latrunculiidae family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponges.

Topoisomerases inhibitory activities and DNA binding properties of 9-methoxycamptothecin from Nothapodytes nimmoniana (J. Graham) Mabberly.

We have reported previously that 9-methoxycamptothecin (MCPT) showed significant antitumor activity in vitro. Here, agarose gel electrophoresis experiments were performed to evaluate MCPT's unwinding ability toward plasmid DNA and inhibitory activities against topoisomerases (Topo) I and II. Binding properties of MCPT to calf thymus DNA (CT-DNA) were evaluated by UV-vis, melting temperature, fluorescence, circular dichroism methodologies and molecular docking technique. Results showed that MCPT at 100 µM inhibited Topo I activity, but had no effect on Topo II. Studies on the binding properties indicated that minor groove binding was the most probable binding mode of MCPT to DNA. The abilities of MCPT to act as Topo I inhibitor and minor groove binding agent may be related to its strong antitumor activity.

Eucalypglobulusals A-J, Formyl-Phloroglucinol-Terpene Meroterpenoids from Eucalyptus globulus Fruits.

Ten new formyl-phloroglucinol-terpene meroterpenoids, eucalypglobulusals A-J (1-10), and ten known analogues were isolated from  Eucalyptus globulus fruits. The structures of 1-10 were determined by spectroscopic analysis, while their absolute configurations were established using calculated and experimental electronic circular dichroism (ECD) spectra. Eucalypglobulusal A was assigned as a new formyl-phloroglucinol-terpene meroterpenoid with a rearranged sesquiterpene skeleton, and an aldol condensation between C-3 and C-5 of the germacrene C moiety was proposed to be a key step in its putative biosynthetic pathway. Eucalypglobulusal F exhibited cytotoxicity against the human acute lymphoblastic cell line (CCRF-CEM) with an IC50 value of 3.3 µM, while eucalypglobulusal A, eucarobustol C, macrocarpal A, macrocarpal B, and macrocarpal D exhibited DNA topoisomerase I (Top1) inhibition. The compounds eucalypglobulusal A and macrocarpal A act as Top1 catalytic inhibitors and delay Top1 poison-mediated DNA double-strand damage.

Screening for anti-proliferative and anti-inflammatory components from Rhamnus davurica Pall. using bio-affinity ultrafiltration with multiple drug targets.

Rhamnus davurica Pall. (R. davurica) has been used as a traditional medicine for many years in China and abroad and shown a wide spectrum of biological activities. Previously, we reported the phytochemical fingerprinting profile of R. davurica, its distinct anti-proliferative activities against HT-29 and SGC-7901 cell lines, and the topoisomerase I (Top I) ligands based on bio-affinity ultrafiltration and HPLC-MS (UF-HPLC-MS). Nevertheless, among the 32 peaks detected in the fingerprinting profile, the common bioactive constituents responsible for the anti-inflammatory and anti-proliferative activities in the extracts remain elusive. To further explore the specific responsible components for their diversified activities and their potential action targets/mechanisms, the method based on bio-affinity UF-HPLC-MS using therapeutic targets like Top I and cyclooxygenase 2 (COX-2) was established to rapidly screen and identify the ligands binding to these known target enzymes. As a result, 12 components were revealed as potential Top I ligands along with 11 components as potential COX-2 ligands, where several components were revealed to possess both activities. Further validations of these bioactive components have also been conducted and confirmed their highlighted activities. This integrated method of UF-HPLC-MS exhibits high efficiency in rapidly screening for multi-target bioactive components responsible for multiple pharmacological effects from the complex natural products and could be very useful to explain the complex action mechanisms of herb medicines in a complex multi-component and multi-target mode at the molecular level. Graphical abstract Schematic diagram of UF-HPLC-MS assay to screen for Top I and COX-2 ligands. The principle of the assay usually involves the following steps: incubation, ultrafiltration, and identification.

From Lead to Drug Utilizing a Mannich Reaction: The Topotecan Story.

Natural products are a rich source of bioactive compounds, and numerous natural compounds have found application in cancer chemotherapy. However, unfavorable physicochemical properties often prevent the use of the original natural product as a drug. A prominent example is camptothecin from the Chinese tree Camptotheca acuminata, which shows extraordinary cytotoxic activity based on a specific molecular mode of action (inhibition of human topoisomerase I). Due to its extremely poor solubility, the original natural product cannot be used as a drug. The marketed drug topotecan was developed from this lead structure by semi-synthesis utilizing a Mannich aminomethylation as the crucial step. In this review, the long-distance run leading to this drug and further perspectives are summarized.

Antiproliferative activities of Amaryllidaceae alkaloids from Lycoris radiata targeting DNA topoisomerase I.

Crude Amaryllidaceae alkaloids (AAs) extracted from Lycoris radiata are reported to exhibit significant anti-cancer activity. However, the specific alkaloids responsible for the pharmacodynamic activity and their targets still remain elusive. In this context, we strived to combine affinity ultrafiltration with topoisomerase I (Top I) as a target enzyme aiming to fish out specific bioactive AAs from Lycoris radiata. 11 AAs from Lycoris radiata were thus screened out, among which hippeastrine (peak 5) with the highest Enrichment factor (EF) against Top I exhibited good dose-dependent inhibition with IC50 at 7.25 ± 0.20 µg/mL comparable to camptothecin (positive control) at 6.72 ± 0.23 µg/mL. The molecular docking simulation further indicated the inhibitory mechanism between Top I and hippeastrine. The in vitro antiproliferation assays finally revealed that hippeastrine strongly inhibited the proliferation of HT-29 and Hep G2 cells in an intuitive dose-dependent manner with the IC50 values at 3.98 ± 0.29 µg/mL and 11.85 ± 0.20 µg/mL, respectively, and also induced significant cellular morphological changes, which further validated our screening method and the potent antineoplastic effects. Collectively, these results suggested that hippeastrine could be a very promising anticancer candidate for the therapy of cancer in the near future.

Cuminaldehyde from Cinnamomum verum Induces Cell Death through Targeting Topoisomerase 1 and 2 in Human Colorectal Adenocarcinoma COLO 205 Cells.

Cinnamomum verum, also called true cinnamon tree, is employed to make the seasoning cinnamon. Furthermore, the plant has been used as a traditional Chinese herbal medication. We explored the anticancer effect of cuminaldehyde, an ingredient of the cortex of the plant, as well as the molecular biomarkers associated with carcinogenesis in human colorectal adenocarcinoma COLO 205 cells. The results show that cuminaldehyde suppressed growth and induced apoptosis, as proved by depletion of the mitochondrial membrane potential, activation of both caspase-3 and -9, and morphological features of apoptosis. Moreover, cuminaldehyde also led to lysosomal vacuolation with an upregulated volume of acidic compartment and cytotoxicity, together with inhibitions of both topoisomerase I and II activities. Additional study shows that the anticancer activity of cuminaldehyde was observed in the model of nude mice. Our results suggest that the anticancer activity of cuminaldehyde in vitro involved the suppression of cell proliferative markers, topoisomerase I as well as II, together with increase of pro-apoptotic molecules, associated with upregulated lysosomal vacuolation. On the other hand, in vivo, cuminaldehyde diminished the tumor burden that would have a significant clinical impact. Furthermore, similar effects were observed in other tested cell lines. In short, our data suggest that cuminaldehyde could be a drug for chemopreventive or anticancer therapy.

Discovery of a novel anti-cancer agent targeting both topoisomerase I and II in hepatocellular carcinoma Hep 3B cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

Cinnamomum verum has been used as a traditional Chinese herbal medicine. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, in hepatocellular carcinoma Hep 3B cells. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by an up-regulation of pro-apoptotic bax and bak genes and down-regulation of anti-apoptotic bcl-2 and bcl-XL genes, mitochondrial membrane potential loss, cytochrome c release, activation of caspase 3 and 9, increase in the DNA content in sub G1, and morphological characteristics of apoptosis. 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments (VAC), suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against Hep 3B cells is accompanied by downregulations of NF-κB binding activity, inflammatory responses involving COX-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and VAC. Our data suggest that 2-MCA could be a potential agent for anticancer therapy.

Versixanthones A-F, Cytotoxic Xanthone-Chromanone Dimers from the Marine-Derived Fungus Aspergillus versicolor HDN1009.

Six unusual xanthone-chromanone dimers, versixanthones A-F (1-6), featuring different formal linkages of tetrahydroxanthone and 2,2-disubstituted chroman-4-one monomers, were isolated from a culture of the mangrove-derived fungus Aspergillus versicolor HDN1009. The absolute configurations of 1-6, representing the central and axial chirality elements or preferred helicities, were established by a combination of X-ray diffraction analysis, chemical conversions, and TDDFT-ECD calculations. The interconversion of different biaryl linkages between 1 and 4 and between 2 and 3 in DMSO by a retro-oxa-Michael mechanism provided insight into the formation of the xanthone-chromanone dimers and supported the assignments of their absolute configurations. Compounds 1-6 exhibited cytotoxicities against the seven tested cancer cell lines, with the best IC50 value of 0.7 µM. Compound 5 showed further inhibitory activity against topoisomerase I.

Cytotoxic Indolocarbazoles from Actinomadura melliaura ATCC 39691.

Actinomadura melliaura ATCC 39691, a strain isolated from a soil sample collected in Bristol Cove, California, is a known producer of the disaccharide-substituted AT2433 indolocarbazoles (6-9). Reinvestigation of this strain using new media conditions led to >40-fold improvement in the production of previously reported AT2433 metabolites and the isolation and structure elucidation of the four new analogues, AT2433-A3, A4, A5, and B3 (1-4). The availability of this broader set of compounds enabled a subsequent small antibacterial/fungal/cancer SAR study that revealed disaccharyl substitution, N-6 methylation, and C-11 chlorination as key modulators of bioactivity. The slightly improved anticancer potency of the newly reported N-6-desmethyl 1 (compared to 6) contrasts extensive SAR of monoglycosylated rebeccamycin-type topoisomerase I inhibitors where N-6 alkylation has contributed to improved potency and ADME. Complete 2D NMR assignments for the known metabolite BMY-41219 (5) and (13)C NMR spectroscopic data for the known analogue AT2433-B1 (7) are also provided for the first time.

Fused aryl-phenazines: scaffold for the development of bioactive molecules.

Fused aryl phenazine derivatives (benzo[a]phenazine, pyrido[a]phenazine, benzo[a]phenazine diones, tetrahydropyrido[a]phenazine (dermacozines), etc) are important heterocyclic compounds, which exhibit various pharmacological activities, prominently in cancer cell lines. These compounds significantly intercalate between DNA base pairs and inhibit the activities of topoisomerase I and II enzymes (Topo I and II). XR11576, XR5944, NC-190 and NC-182 belong to phenazine/fused aryl phenazine category and are under clinical studies. Several fused aryl phenazine dione compounds such as pyridazino[4,5-b]phenazine-5,12-diones, 6,11-dihydro-pyrido[2,3-b]phenazine-6,11-diones, 6,11-dihydrobenzo[2,3-b]phenazine-6,11-diones, tetrahydropyrido[a]phenazine, etc possessed anticancer activities on various cancer cell lines. Benzo[a]phenazine diimine and various other fused aryl phenazine compounds form coordination complex with the metal ions (Ru, Rh, Zn and Pt) that intercalate with the DNA and are used for the treatment of cancer. These molecules have influence on MDR cancer cells and serve as anticancer agents in MDR cancer cells. The structure activity relationship of the fused aryl phenazine derivatives revealed that the occurrence of four or more nitrogen atoms in the compounds has better anticancer activity than those molecules with less number of nitrogen atoms. Phenazine antibiotics derived from marine microbes are used for the treatment of microbial and worm diseases. Recent patents on these scaffolds showed that the benzo[a]phenazine derivatives have inhibitory activity on topoisomerase enzymes (Topo I and II) and that act as anticancer agents.

Inhibition of DNA topoisomerases I and II of compounds from Reynoutria japonica.

Three anthraquinones (1, 2 and 4), three stilbenes (5, 6 and 7) and 3,5-dihydroxybenzyl alcohol (3) were isolated from Reynoutria japonica. Their structures were identified as emodin (1), emodin-8-O-ß-D-glucoside (2), 3,5-dihydroxybenzyl alcohol (3), citreorosein (4), cis-resveratrol (5), trans-resveratrol (6) and trans-resveratrol-5-O-ß-D-glucopyranoside (7) by comparing their physicochemical and spectral data with published data. Compound 3 was isolated for the first time from the Polygonaceae family. Among the purified compounds, 3 showed more potent inhibitory activity against topoisomerase I (IC50: 4 µM) than camptothecin, as the positive control (IC50: 18 µM). Compounds 3, 4, 5, 6 and 7 showed stronger inhibitory activities toward DNA topoisomerase II (IC50: 0.54, 14, 15, 0.77 and 3 µM, respectively) than the positive control, etoposide (IC50: 44 µM). Compounds 1 and 4 displayed weak cytotoxicities against human lung cancer (A549), ovarian cancer (SK-OV-3), human liver hepatoblastoma (HepG2) and colon adenocarcinoma (HT-29) cell lines.

Abietane diterpenes induce cytotoxic effects in human pancreatic cancer cell line MIA PaCa-2 through different modes of action.

Abietane diterpenes, especially those containing quinone moieties, are often reported to have cytotoxic effects on cancer cell lines. They deserve greater attention because several cancer chemotherapeutic agents also possess the quinone structural feature. To date, very little is known about their cytotoxic molecular modes of action. In the present study, five diterpenes, 7 alpha-acetoxyroyleanone, horminone, royleanone, 7-ketoroyleanone and sugiol which have been previously isolated from the medicinal plant Peltodon longipes were shown to possess cytotoxic activity against the human pancreatic cancer cell line MIA PaCa-2. 7 alpha-Acetoxyroyleanone, horminone and royleanone were demonstrated to possess alkylating properties using the nucleophile 4-(4-nitrobenzyl)pyridine. However, no clear correlation between the alkylating properties and cytotoxicity of these diterpenes was observed. Furthermore, the relaxation activity of human DNA topoisomerases I and II was found to be influenced by these compounds, with 7-ketoroyleanone and sugiol being the most active. These two diterpenes preferentially inhibited topoisomerase I and exhibited lower IC(50) values than the classical topoisomerase I inhibitor camptothecin. Molecular docking studies revealed possible interactions of diterpenes with topoisomerase I, indicating that these compounds do not form the drug-enzyme-DNA covalent ternary complex as observed with camptothecin. A binding pocket located at the surface of the DNA-interaction site was proposed. Moreover, the ability of the five diterpenes to generate DNA-strand breaks in single cells was confirmed using the alkaline comet assay. As expected, these diterpenes also influenced cell cycle progression and arrested cells in different phases of the cell cycle, primarily the G1/G0 and S-phases. Interestingly, the diterpenes only exhibited a slight ability to induce apoptotic cell death and failed to generate intracellular reactive oxygen species. These results provide additional understanding of the cytotoxic effects of abietane diterpenes. Depending on their functional groups, we propose that abietane diterpenes utilise different mechanisms to induce cell death.

Constituents with DNA topoisomerases I and II inhibitory activity and cytotoxicity from the roots of Rubia cordifolia.

Activity-directed isolation of the ethyl acetate fraction from the roots of Rubia cordifolia resulted in the identification of a new anthraquinone, 1,3,6-trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3- O- α- L-rhamnopyranosyl-(1 → 2)- ß-D-(6'-O-acetyl)-glucopyranoside (1), two new dihydronaphtoquinones, 1,4-dihydroxy-2-carbomethoxy-3-prenylnaphthalene-1-O- ß-D-glucopyranoside (2) and mollugin-1-O- ß- D-glucopyranoside (3), and a new monoterpenoid, 3 R,3a S,4 R,6a R-3,4,6-tris(hydroxymethyl)-3,3a,4,6a-tetrahydro-2 H-cyclopenta[ B]furan-2-one (4), together with nine known compounds (5-13). The structures of these compounds were elucidated on the basis of spectroscopic evidence. In addition, their DNA topoisomerases I and II inhibitory activity and cytotoxicity were measured.

ALCAPs induce mitochondrial apoptosis and activate DNA damage response by generating ROS and inhibiting topoisomerase I enzyme activity in K562 leukemia cell line.

Endemic Alkanna cappadocica was used to isolate novel antitumor molecules from Turkish landscapes in our previous studies. In this study, deoxyalkannin (ALCAP1), ß,ß-dimethylacrylalkannin (ALCAP2), acetylalkannin (ALCAP3), and alkannin (ALCAP4) as well as the novel isolated compounds 5-methoxydeoxyalkannin (ALCAP5), 8-methoxydeoxyalkannin (ALCAP6), 5-methoxyacetylalkannin (ALCAP7), 5-methoxy-ß,ß-dimethylacrylalkannin (ALCAP8) were characterized. The topoisomerase I (topo I) inhibitory activity of ALCAPs was investigated using in vitro plasmid relaxation assay and found that ALCAP2, 3, 4 and 7 were potent inhibitors at 2-6µM concentrations. Further, DNA damage response to ALCAP treatments was also studied by measuring the H2AX((S139)) and ATM((S1981)) phosphorylations. ALCAP2, 7 and 8 induced the DNA damage and apoptosis, consistently resulted in PARP cleavage at nanomolar concentrations in K562 leukemia cells. Moreover, when the free radical (ROS) generating capacity of the compounds was studied by 2',7'-dichlorofluorescein-diacetate assay using flow cytometry, we found that a known antioxidant N-acetyl-cysteine almost completely abrogated the H2AX((S139)) phosphorylations and the caspase 3 cleavage and activation. Thus, γH2AX((S139)) foci formation remained higher than the control, and an increase in CHK2((T68)) phosphorylation was observed by ALCAP2 and 7 treatments suggested that, these compounds can be potential therapeutics against tumor cell growth because of their unique DNA damaging abilities additional to enzyme inhibition similar to those of doxorubicin.

Secalonic acid D as a novel DNA topoisomerase I inhibitor from marine lichen-derived fungus Gliocladium sp. T31.

CONTEXT: DNA topoisomerase I (topo I) is an essential enzyme which regulates the conformational changes in DNA topology by cleaving and rejoining DNA strands during normal cell growth. The inhibitors of topo I represent a major class of anticancer drugs. In our projects to isolate new anticancer agents from marine-derived fungi, secalonic acid D (SAD) with inhibitory activity on topo I was isolated from the fermentation broth of marine lichen-derived fungus Gliocladium sp. T31, which was collected from marine sediments in South Pole. OBJECTIVE: The inhibitory activity of SAD on topo I was investigated for the first time. MATERIALS AND METHODS: The inhibitory effect of SAD on topo I was determined via in vitro supercoil relaxation assays and electrophoretic mobility shift assay (EMSA) using plasmid substrate, pBR322. RESULTS: SAD displays a considerable inhibition on topo I in a dose-dependent manner with the minimum inhibitory concentration (MIC) of 0.4 µM. Unlike the prototypic DNA topo I poison camptothecin (CPT), SAD inhibits the binding of topo I to DNA but does not induce the formation of topo I-DNA covalent complexes. DISCUSSION AND CONCLUSION: SAD is an excellent topo I inhibitor and thus a significantly potential anticancer candidate.

3-O-(E)-p-coumaroyl tormentic acid from Eriobotrya japonica leaves induces caspase-dependent apoptotic cell death in human leukemia cell line.

Eleven triterpene acids, 1-11, isolated from the leaves of Eriobotrya japonica, were evaluated for inhibition of DNA topoisomerase (Topo) I and cytotoxicity against human leukemia (HL60) and melanoma cell lines (CRL1579). Among the compounds tested, four compounds, δ-oleanolic acid (4), ursolic acid (5), 3-O-(E)-p-coumaroyl tormentic acid (8), and betulinic acid (10), exhibited potent Topo I inhibitory activity (IC(50) 20.3-36.5 µM) and cytotoxicity against HL60 (EC(50) 5.0-8.1 µM). Upon assessing the apoptosis-inducing activity in HL60 cells, compound 8 exhibited induction of apoptosis detected by the observation of DNA fragmentation and membrane phospholipid exposure in flow cytometry. Western blot analysis showed that compound 8 markedly reduced the levels of procaspases-3 and 9, while being increased the levels of cleaved caspases-3 and 9. On the other hand, compound 8 exerted almost no influence on the expression of caspase-8. In addition, compound 8 increased significantly Bax/Bcl-2 ratio and activated caspase-2. These results suggested that compound 8 induced apoptotic cell death in HL60 via mainly mitochondrial pathway by, at least in part, Topo I inhibition. Therefore, compound 8 may be promising lead compound for developing an effective drug for treatment of leukemia.