6-Acetonyldihydrochelerythrine Is a Potent Inducer of Apoptosis in HCT116 and SW620 Colon Cancer Cells.

6-Acetonyldihydrochelerythrine (1), a benzophenanthridine alkaloid, isolated from the methanol extract of Zanthoxylum capense, displayed potent cytotoxic activity in human HCT116 and SW620 colon carcinoma cells, to a higher extent than 5-fluorouracil (5-FU), the cornerstone chemotherapeutic agent in colon cancer. Cytotoxicity of 1 was evaluated by MTS, lactate dehydrogenase (LDH), and Guava ViaCount assays. Interestingly, 1 significantly induced cytotoxicity in both cell lines, leading to a significant increase in LDH release, as compared to 5-FU. Further, Guava ViaCount flow cytometry assays demonstrated that 1 significantly increased cell death, as shown by the presence of a significantly higher population of apoptotic cells in both cell lines, as compared to cells exposed to 5-FU. Furthermore, evaluation of nuclear morphology by Hoechst staining of 1-treated HCT116 and SW620 cells confirmed flow cytometry results, demonstrating a marked induction of apoptotic cell death by 1, again to a further extent than that elicited by 5-FU. In addition, immunoblot analysis to ascertain the molecular events triggered by 1 exposure was performed. The results show that 1 exposure reduced the steady-state expression and activation of the pro-survival proteins ERK5 and Akt and increased the steady-state expression of p53 in both HCT116 and SW620 cells. Changes in ERK5 or Akt activation can be ascertained by evaluating the ratio of p-ERK5/ERK5 or p-Akt/Akt. In addition, exposure to 1 reduced expression of XIAP, Bcl-XL, and Bcl-2, while increasing the cleavage of poly(ADP-ribose) polymerase in both cell lines. Collectively, the data indicate that 6-acetonyldihydrochelerythrine (1) is a potent inducer of apoptosis in HCT116 and SW620 cell lines, highlighting its potential relevance in colon cancer.

Anti-inflammatory guaiane-type sesquiterpenes from the fruits of Pittosporum undulatum.

Two unprecedented guaiane-type sesquiterpene glycosides (undulatumosides A and B) were isolated by bioassay-guided fractionation from the MeOH extract of Pittosporum undulatum fruits, along with six known compounds, including the guaiane isomers 5-guaien-11-ol and 4-guaien-11-ol. The structures of the compounds were established as 4-guaiene-11-O-ß-d-(3'-angeloxy-6'-deoxy)-glucopyranoside and 1(5)-guaiene-11-O-ß-d-(3'-angeloxy-6'-deoxy)-glucopyranoside by spectroscopic methods, including 1D and 2D homo- and heteronuclear NMR experiments (COSY, HSQC, HMBC and NOESY), and HR-mass spectrometry. P. undulatum is a highly invasive weed that often outcompetes other plants, yet its fruits have become a traditional anti-inflammatory medicine in Azores. Therefore, aiming to investigate the claimed properties, the in vitro anti-inflammatory activity of guaiane-type sesquiterpenes was evaluated by analyzing their inhibitory effects on chemical mediators released by the LPS activated RAW 264.7 murine macrophages cell line. In addition, the cytotoxicity of these compounds was also evaluated in this cell line. Undulatumoside A, 5-guaien-11-ol and 4-guaien-11-ol displayed anti-inflammatory activity with IC50 values of 16.4, 8.1 and 7.2µM, respectively, comparable to that of the positive control, indomethacin (IC50=18.2 µM), with no cytotoxic effects (IC50 ≥ 198 µM). Furthermore, the same set of compounds was also assessed for anti-proliferative activity in lung large cell carcinoma COR-L23 and amelanotic melanoma C32 cells.

Monoterpene bisindole alkaloids, from the African medicinal plant Tabernaemontana elegans, induce apoptosis in HCT116 human colon carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Tabernaemontana elegans is a medicinal plant used in African traditional medicine to treat several ailments including cancer. The aims of the present study were to identify anti-cancer compounds, namely apoptosis inducers, from Tabernaemontana elegans, and hence to validate its usage in traditional medicine. METHODS AND MATERIALS: Six alkaloids, including four monomeric indole (1-3, and 6) and two bisindole (4 and 5) alkaloids, were isolated from the methanolic extract of Tabernaemontana elegans roots. The structures of these compounds were characterized by 1D and 2D NMR spectroscopic and mass spectrometric data. Compounds 1-6 along with compound 7, previously isolated from the leaves of the same species, were evaluated for in vitro cytotoxicity against HCT116 human colon carcinoma cells by the MTS metabolism assay. The cytotoxicity of the most promising compounds was corroborated by Guava-ViaCount flow cytometry assays. Selected compounds were next studied for apoptosis induction activity in HCT116 cells, by evaluation of nuclear morphology following Hoechst staining, and by caspase-3 like activity assays. RESULTS: Among the tested compounds (1-7), the bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were found to be cytotoxic to HCT116 cells at 20 µM, with compound 5 being more cytotoxic than the positive control 5-Fluorouracil (5-FU), at a similar dose. In fact, even at 0.5 µM, compound 5 was more potent than 5-FU. Compounds 4 and 5 induced characteristic patterns of apoptosis in HCT116 cancer cells including, cell shrinkage, condensation, fragmentation of the nucleus, blebbing of the plasma membrane and chromatin condensation. Further, general caspase-3-like activity was increased in cells exposed to compounds 4 and 5, corroborating the nuclear morphology evaluation assays. CONCLUSIONS: Bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were characterized as potent apoptosis inducers in HCT116 human colon carcinoma cells and as possible lead/scaffolds for the development of anti-cancer drugs. This study substantiates the usage of Tabernaemontana elegans in traditional medicine to treat cancer.

Apoptosis inducing activity of benzophenanthridine-type alkaloids and 2-arylbenzofuran neolignans in HCT116 colon carcinoma cells.

Thirteen compounds belonging to different classes of alkaloids (1-9) and lignans (10-13), isolated from the methanol extract of roots of the African medicinal plant Zanthoxylum capense, were assayed for their ability as apoptosis inducers in HCT116 colon carcinoma cells. The cytotoxicity of these compounds was evaluated in HCT116 colon carcinoma cells by the MTS assay. Out of the tested compounds, three benzophenanthridine alkaloids (1, 4, and 7), a dibenzyl butyrolactone lignan (10), and two 2-arylbenzofuran neolignans (12 and 13) displayed significant cytotoxicity to HCT116 cells, confirmed by the Guava ViaCount viability assay. The selected compounds (1, 4, 7, 10, 12, and 13) were further tested for apoptosis induction activity in HCT116 cells, by evaluation of nuclear morphology following Hoechst staining, and by caspase-3 like activity assays. Morphologic evaluation of HCT116 nuclei following Hoechst staining and fluorescence microscopy revealed that compounds 1, 4, 7, 10, 12, and 13 induced apoptosis in HCT116 colon carcinoma cells, producing similar, or higher, apoptosis levels when compared with 5-fluorouracil (5-FU), the cornerstone cytotoxic used in colon cancer treatment for several decades. In fact, HCT116 cells developed morphological changes characteristic of apoptosis, including chromatin condensation, nuclear fragmentation and formation of apoptotic bodies. Importantly, compounds 4 and 13 at 20 µM were the most promising in this study, inducing respectively ∼11- and 7-fold increases in apoptotic cells as compared to vehicle control, whereas 5-FU increased apoptosis by ∼2-fold. Apoptosis induction for compounds 4 and 13 was further confirmed by caspase-3-like activity assays, which showed respectively ∼2- and 1.5-fold increases in caspase-3-like activity compared to vehicle control. These results suggested that specific benzophenanthridine alkaloids and 2-arylbenzofuran neolignans isolated from Zanthoxylum capense show strong anticancer activity in HCT116 colon carcinoma cells.

Dibenzylbutane- and butyrolactone-type lignans as apoptosis inducers in human hepatoma HuH-7 cells.

Seven lignans, previously isolated from Pycnanthus angolensis or obtained by derivatization, namely the dibenzylbutane-type lignans threo-4,4'-dihydroxy-3-methoxylignan (1), 4'-hydroxy-3,3',4-trimethoxylignan (2), (-)-dihydroguaiaretic acid (3), 3,3',4,4'-tetramethoxylignan (4), 4,4'-diacetyl-3,3'-dimethoxylignan (5), heliobuphthalmin (6) and the butyrolactone lignan hinokinin (7), were evaluted for their ability as apoptosis inducers in human hepatoma HuH-7 cells. Cell viability assays, morphological evaluation of apoptosis and enzymatic analyses of caspase activity in HuH-7 cells were carried out. Using the lactate dehydrogenase lactate dehydrogenase (LDH) assay, it was demonstrated that the lignans (1-7) tested significantly reduced viability of HuH-7 cells. Morphologic evaluation of HuH-7 cells using Hoechst staining and fluorescence microscopy revealed that lignans 1-7 were strong inducers of apoptosis. In fact, HuH-7 cells developed morphological changes of apoptosis, including chromatin condensation, nuclear fragmentation and formation of apoptotic bodies. However, lignans 2 and 7 were the most promising compounds in this study, inducing 2.4- and 2.5-fold increases in apoptotic cells as compared to controls. Caspase-3-like activity assays confirmed the morphologic data.

Isoflavones as apoptosis inducers in human hepatoma HuH-7 cells.

Nine flavonoids isolated from the ethyl acetate extract of Pycnanthus angolensis were assayed for their potential apoptosis induction activities in human hepatoma HuH-7 cells. These flavonoids include eight isoflavones, namely irilone (1), tectorigenine (2), formononetin (3), genistein (4), 2'-hydroxybiochanin A (5), mixture of biochanin A (6) and prunetin (7), and 4',7-dihydroxy-2'-methoxyisoflavan (8), and the flavanone liguiritigentin (9). Their chemical structures were characterized by spectroscopic methods including 2D NMR experiments. Methodology for cell death detection included the LDH assay, Hoechst staining, TUNEL staining and general caspase-3-like activity assay. The compounds tested showed higher apoptosis induction profiles in HuH-7 cells compared with the control. Caspase activity assays confirmed the apoptosis inducing activity of these flavonoids.

Cucurbitane-type triterpenoids from the African plant Momordica balsamina.

Phytochemical investigation of the aerial parts of Momordica balsamina led to the isolation of five new cucurbitane-type triterpenoids (1-5) and two known analogues (6, 7). Their structures were elucidated on the basis of spectroscopic methods including 2D NMR experiments (COSY, HMQC, HMBC, and NOESY). The new compounds feature unusual oxidation patterns in the cucurbitane skeleton, such as at C-29 (1-3) and C-12 (4, 5). Compounds 1-4, 6, and 7 were evaluated for in vitro cytotoxicity against human breast cancer cells (MCF-7), using the MTT assay.

Induction of apoptosis in HuH-7 cancer cells by monoterpene and beta-carboline indole alkaloids isolated from the leaves of Tabernaemontana elegans.

Three known (1-3) and a novel (4) monoterpene indole alkaloids have been isolated from the methanol extract of leaves of Tabernaemontana elegans and their structures were elucidated by a series of spectroscopic experiments, involving NMR, MS, UV, and IR techniques. The isolated monoterpene indole alkaloids along with previously described beta-carbolines (5-7) from the same specimen were studied for their apoptosis induction activity in human hepatoma HuH-7 cells. Methodology for apoptosis induction studies included cell viability assays, nuclear morphology assessments, and general caspase-3-like activity assays. The monoterpene indole alkaloids, tabernaemontanine (1) and vobasine (3) showed the most promising apoptosis induction profile in HuH-7 cells.

Tabernines A-C, beta-carbolines from the leaves of Tabernaemontana elegans.

Three novel beta-carboline indole alkaloids (1-3) have been isolated from a MeOH extract of the leaves of Tabernaemontana elegans. The structures were established by means of spectroscopic techniques including 2D NMR experiments. Compounds 1 and 2 contain a two-carbon unit, attached to a structurally related beta-carboline skeleton, as part of an additional six-membered ring in 1 and a seven-membered ring in 2. To the best of our knowledge, this is the first report of beta-carboline indole alkaloids from the genus Tabernaemontana. Compounds 1-3 were evaluated for their ability to modulate multidrug resistance in mouse lymphoma cell lines. Compounds 1 and 3 exhibited a weak activity.

Renierosides, cerebrosides from a marine sponge Haliclona (Reniera) sp.

Guided by the brine shrimp lethality assay, eight new cerebrosides (1-8) have been isolated from an extract of the marine sponge Haliclona (Reniera) sp. A novel feature of these cerebrosides was the presence of unprecedented amide-linked long-chain fatty acid moieties. The planar structures of the cerebrosides (1-8) were established by 1D and 2D NMR spectroscopic techniques, mass spectrometric analyses, and chemical degradation methods. The isolated compounds did not display cytotoxicity to a panel of five human solid tumor cell lines.

5,6:8,9-diepoxy and other cytotoxic sterols from the marine sponge Homaxinella sp.

Four new (1, 2, 4, and 5) and 14 known (3 and 6-18) polyoxygenated sterols have been isolated from the MeOH extract of the marine sponge Homaxinella sp. by bioactivity-guided fractionation. The planar structures of the sterols were established by 1D and 2D NMR and MS spectroscopic analysis. 5,6:8,9-Diepoxy sterols (1-3) were isolated from a marine organism for the first time. The isolated sterols were tested against a panel of five human solid tumor cell lines and exhibited varying degrees of cytotoxicity.

New fatty acid derivatives from Homaxinella sp., a marine sponge.

Fractionation of the MeOH extract of Homaxinella sp., a marine sponge, led to the isolation of a sodium salt of a new brominated FA (1), two new MG (2 and 4), and a new lysoPC (6). The geometry of the double bonds in 1 and 2 was defined by comparison of the NMR chemical shifts of the allylic carbons, nuclear Overhauser effect spectroscopy correlations of the allylic protons, and coupling constants of the vinylic protons with those reported. Evidence mainly from NMR and MS analyses established the planar structures of the compounds. Compounds 1, 2, 4, and 6 were evaluated for cytotoxicity against a panel of five human solid tumor cell lines. Only compound 1 showed moderate activity.

Cytotoxic sterol derivatives from a marine sponge Homaxinella sp.

A bioactivity-guided fractionation of a marine sponge Homaxinella sp. has led to the isolation of three new (1-3) highly degraded sterols and four new 6-O-alkylated (6-9) sterols, along with known sterol derivatives. The degraded sterols (1-5) belong to the class incisterols, previously isolated from the marine sponge Dictyonella incisa. Mainly NMR and MS spectroscopic analyses established the gross structures of the new compounds. The relationship between the stereoisomerism of the side chain and HPLC retention time has also been discussed. The compounds were tested against a panel of five human solid tumor cell lines, and especially the degraded sterols (1-4) displayed significant cytotoxicity.

New cytotoxic metabolites from a marine sponge Homaxinella sp.

Three new butenolides (1-3), a new cyclopentenone derivative (4), and a known alcohol (5) were isolated from a marine sponge Homaxinella sp. by bioactivity-guided fractionation. The planar structures were established on the basis of NMR and MS analyses. The stereochemistry of the butenolides and cyclopentenone derivative was defined on the basis of optical rotation and CD spectroscopy. The compounds were tested for cytotoxicity against a panel of five human solid tumor cell lines and displayed marginal to significant activity.

New cytotoxic sesterterpenoids and norsesterterpenoids from two sponges of the genus Sarcotragus.

New norsesterterpenoids (3 and 4), a sesterterpenoid (6), pyrroloterpenoids (7-10), and a stereoisomer of kurospongin (5) were isolated, along with known furanosesterterpenes (11-15), from two marine sponges of the genus Sarcotragus. The gross structures were established on the basis of NMR and MS analysis. The stereochemistry was defined by combined use of NMR and CD spectroscopy. The compounds were evaluated for cytotoxicity against five human tumor cell lines and were found to exhibit marginal to moderate activity.

New lysophosphatidylcholines and monoglycerides from the marine sponge Stelletta sp.

Two new lysophosphatidylcholines (1, 2) and four new monoglycerides (5-8) were isolated from the marine sponge Stelletta sp. by bioactivity-guided fractionation. The planar structures of the new compounds were established on the basis of NMR and MS analyses. The stereochemistry was defined by comparison of the optical rotation. The compounds were evaluated for cytotoxicity against a small panel of five human tumor cell lines.