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The novel cyclopenta[b]benzofuran, silvestrol, isolated from the fruits and twigs of Aglaia foveolata, has been found to exhibit very potent in vitro cytotoxic activity against several human cancer cell lines. Furthermore, it was active in the in vivo P388 murine leukemia model. In this study, the mechanism of cytotoxicity mediated by silvestrol in the LNCaP (hormone-dependent human prostate cancer) cell line was investigated. Silvestrol induced an apoptotic response, disrupted the mitochondrial trans-membrane potential and caused cytochrome c release into the cytoplasm. Immunoblot analysis indicated that, at the protein level, silvestrol produced an increase of Bcl-xl phosphorylation with a concomitant increase of bak. Furthermore, caspase-2, -9 and -10 appeared to be involved in silvestrol-mediated apoptosis. In contrast, the involvement of caspase-3 and -7 was not detected, either by immunoblot or caspase-3/-7-like activity analysis, indicating that these pathways do not play a crucial role in silvestrol-induced apoptosis. To investigate the relative contribution of the caspases, inhibition of apoptosis with four different cell-permeable inhibitors was studied (Boc-D-Fmk, Z-VDVAD-FMK Z-LEHD-FMK and Z-AEVD-FMK). Only the general caspase inhibitor, Boc-D-Fmk, completely inhibited the formation of apoptotic bodies. In contrast, caspase-2 and caspase-9 selective inhibitors induced about a 40% reduced apoptotic response, whereas the caspase-10 selective inhibitor caused about a 60% reduction in apoptosis compared to silvestrol only treated cells. Taken together, the studies described herein demonstrate the involvement of the apoptosome/mitochondrial pathway and suggest the possibility that silvestrol may also trigger the extrinsic pathway of programmed cell death signaling in tumor cells.

As previously reported, silvestrol, a rocaglate derivative isolated from Aglaia foveolata, has similar potency to paclitaxel and camptothecin against cultured human cancer cells. Furthermore, silvestrol can inhibit cancer cell growth in mice without noticeable toxicity when administered up to 5 mg/kg body weight (the highest dose tested). The purpose of the current study was to evaluate the mechanism of silvestrol's cytotoxicity in human prostate cancer cells (LNCaP). The molecular signature induced in LNCaP cells by silvestrol was evaluated using microarray analysis. The results revealed that 20 apoptosis and cell cycle related genes were significantly altered in LNCaP cells exposed to silvestrol. These included UBL-3, p21 and p300, which were up-regulated, and p53, which was down-regulated. Since p53 expression is governed primarily at the level of translation, p53 was also evaluated by Western blot. Silvestrol caused a dose-dependent decrease in p53 protein within 30 min of exposure with no p53 detectable after 6 h. Down-regulation of p53 by silvestrol was associated with down-regulation of MDM2 and not prevented by lactacystin suggesting that silvestrol-induced degradation of p53 is not mediated by the proteasome. A slight decrease in cyclin B was observed within 6 h of silvestrol exposure and phosphatase Cdc25C protein, which activates Cdc2, was also decreased. These data demonstrate that cytotoxicity induced by silvestrol in LNCaP cells is associated with a block in the cell cycle at the G2/M checkpoint and alterations in the expression of genes regulating apoptosis and cell cycle in a manner independent of p53.

Rocaglaol is a cytotoxic cyclopenta[b]benzofuran isolated from the bark of Aglaia crassinervia. It exhibited in vitro cytotoxic activity against Lu1, LNCaP and MCF-7 cells with ED50 values of 13.8, 23.0 and 9.2 nM, respectively. DAPI staining indicated that LNCaP cells treated with rocaglaol underwent apoptosis. In order to determine whether rocaglaol-induced apoptosis is mediated by the mitochondrial pathway, apoptosis-related mitochondrial-associated proteins were studied. Rocaglaol treatment induced Bax expression through 12 to 72 h of exposure, while Bcl-xl expression was slightly decreased through 48 h, and decreased more significantly by 72 h. Cleaved caspase-9 expression was detected at 72 h, and cleaved caspase-7 was increased through 48 to 72 h. Consequently, the large fragment (89 kDa) of PARP resulting from caspase cleavage was detected at 12, 24 and 48 h, and especially at 72 h. Cleaved PARP expression was also detected at 72 h. Since rocaglaol caused dose-dependent G2/M phase arrest of LNCaP cells as indicated by flow cytometric analysis, the protein levels of cell cycle-related genes were measured. Rocaglaol treatment (230 nM) did not change cyclin B after 24- to 60-h treatment. The expression of cdc2 was not changed and phospho-cdc2 (Tyr 15) increased after 36-, 48- and 60-h treatment. In addition, protein phosphatase Cdc25C, which functions as a mitotic activator by dephosphorylation of Cdc2, decreased in a time-dependent manner after rocaglaol treatment. Taken together, these results suggest that rocaglaol is a potent anticancer drug that induces apoptosis of LNCaP cells through the mitochondrial pathway and its G2/M-phase cell cycle arrest is associated with the down-regulation of Cdc25C and the dephosphorylation of Cdc2.

Two anthracenone C-glycosides, alvaradoins E and F, isolated from the leaves of Alvaradoa haitiensis Urb. (Simaroubaceae), were found to have potent inhibitory activities with cultured cancer cells. Using the in vivo hollow fiber model, these compounds demonstrated significant growth inhibition at the i.p. site when tested with KB, LNCaP, and Col2 cells. To determine if these anthracenone C-glycosides mediated anticancer activity through an apoptotic pathway, a series of assays were performed with the 10S isomeric compound, alvaradoin E. With a DAPI assay, treatment of LNCaP cells with alvaradoin E at concentrations of 0.4, 2, 10, or 50 microM for 24 or 48 h showed chromatin condensation, a morphological characteristic of apoptosis. Mitochondrial membrane potential, analyzed with a DiOC6 uptake assay, showed that treatment of LNCaP cells with 0.07, 0.14, 0.28, 0.56, 0.86, and 1.12 microM alvaradoin E for 12 h caused dose-dependent membrane depolarization, another indication of early apoptosis. Also, with an annexin V-FITC assay system, treatment of HL-60 cells with 0.07 microM alvaradoin E for 24 h increased annexin V-FITC binding from 3 to 25.9% (8.6-fold). Finally, with the TUNEL assay system, treatment of HL-60 cells with 1.12 microM alvaradoin E for 32 h increased FITC-dUTP binding from 1.2 to 12.1% (10-fold). These data suggest alvaradoin E is an effective anticancer agent that induces apoptosis. Additional studies to establish clinical utility should be of interest.

P-glycoprotein (Pgp)-mediated drug efflux can yield a multidrug resistance (MDR) phenotype that is associated with poor response to cancer chemotherapy. Pervilleine F, a new tropane alkaloid aromatic ester obtained from a chloroform extract of the roots of Erythroxylum pervillei as the result of bioactivity-guided fractionation, was found to restore the vinblastine sensitivity of cultured multidrug-resistant KB-V1 cells, with an IC50 value of 0.40 microM. Pervilleine F (8 microM) was also able to partially reverse the cross-resistance of KB-V1 cells to the clinically used or experimental anticancer agents actinomycin D (45.1-fold), baccatin III (> 3.4-fold), daunomycin (> 22.5-fold), ellipticine (1.9-fold), mithramycin A (42.5-fold), podophyllotoxin (1.6-fold), paclitaxel (32.2-fold) and vincristine (73.6-fold). While pervilleine F alone at the concentration of 10 microM had no significant effect on the KB-V1 cell cycle, pervilleine F (at concentrations of 0.2, 1, 2, and 8 microM) combined with vinblastine (1 microgram/ml) induced dose-dependent G2/M phase arrest, ranging from 20.2, 51.0, 63.7, to 79.5%, as an indication of the restoration of vinblastine sensitivity. To confirm this activity with an in vivo animal model, KB-V1 cells were placed in hollow fibers and implanted into NCr nu/nu mice. Cell growth was not significantly inhibited when vinblastine or pervilleine F was administered as single agents, but when these two compounds were used in combination, inhibition of up to 64.1% was observed. Equimolar doses of verapamil were less effective. These data suggest that pervilleine F is an effective inhibitor of Pgp and should be further evaluated for clinical utility.

P-Glycoprotein (Pgp) associated multidrug-resistance (MDR) is a significant factor that can lead to the failure of cancer chemotherapy. Several new tropane alkaloid aromatic esters obtained from extracts of Erythroxylum pervillei Baillon (Erythroxylaceae) (1-8) and Erythroxylum rotundifolium Lunan (Erythroxylaceae) (9-12) by means of bioassay-directed fractionation were found to restore vinblastine sensitivity with cultured multidrug-resistant KB-VI cells. With this model, growth was not inhibited by addition of vinblastine (1 microg/ml) to the culture medium, but in combination with tropane alkaloids, inhibition was observed with IC50 values categorized as: low (ranging from 0.17-0.62 microM) for 1 [3alpha-phenylacetoxy-6beta-(3,4,5-trimethaxycinnamoyloxy)-tropane], 3 [3alpha-(3,4,5-trimethoxybenzoyloxy)-6beta-(3,4,5-trimethoxycinnamoyloxy)tropane], 4 [3alpha-(3,4,5-trimethoxybenzoyloxy)-6beta-(3,4,5-trimethoxycinnamoyloxy)-7beta-hydroxytropane], 5 [3alpha,6beta-di-(3,4,5-trimethoxycinnamoyloxy)tropane], 6 [3alpha,6beta-di-(3,4,5-trimethoxycinnamoyloxy)-7beta-hydroxytropane] and 9 [6beta-benzoyloxy-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane]; medium (2.0-3.7 microM) for 2 [3alpha-(3-hydroxyphenylacetoxy)-6beta-(3,4,5-trimethoxycinnamoyloxy)tropane] and 10 [7beta-acetoxy-6beta-benzoyloxy-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane]; or high (9.8 microM) for 11 [6beta-benzoyloxy-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane-7beta-ol]. Compounds 7 (tropane-3alpha,6beta,7beta-triol 3-phenylacetate), 8 (1alphaH, 5alphaH-tropan-3alpha-yl 3,4,5-trimethoxybenzoate) and 12 (6beta-(3,4,5-trimethoxybenzoyloxy)-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane-7beta-ol) were not active. Among the active compounds, 1 and 3-6 were further tested with drug-resistant CEM/VLB100 cells. In the presence of modulator, sensitivity to vinblastine increased by 50-5,000-fold. Treatment of KB-V1 cells with 1 or 3-6 enhanced the intracellular accumulation of fluorescence dye (rhodamine 123). Visualization by confocal microscopy confirmed the intracellular accumulation of rhodamine 123 in drug-resistant KB-V1 cells was significantly less than drug-sensitive KB-3 cells, while treatment of KB-V1 cells with 10 microM 4 significantly increased intracellular accumulation. The molecular characteristics of the isolates were then determined and compared with their potential to reverse drug resistance. ClogP and molar refractivity were weakly correlated with their potential to reverse MDR