Antitumor activity and mechanism of action of the cyclopenta[b]benzofuran, silvestrol

Abstract

Background: Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model.

Methodology/principal findings: Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs.

Conclusions/significance: Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.

Figure 1. Structure-activity relationship analysis of cyclopenta[b]benzofurans.

A. Chemical structure of cyclopenta[b]benzofurans tested in this study. B. Effect of cyclopenta[b]benzofurans on cap- and HCV-mediated translation initiation. Krebs-2 translation extracts were programmed with FF/HCV/Ren mRNA and vehicle (MeOH) or 50 µM compound. The relative activity (compared to DMSO controls) from 3 independent translation reactions is presented along with the standard error of the mean. [The partial inhibition of HCV-mediated translation has been previously documented .] C. Dose–dependent inhibition of translation by flavaglines in RRL extracts. Extracts were programmed with FF/HCV/Ren mRNA and firefly luciferase values determined and set relative to translations containing vehicle alone. The values obtained are the average of 2 experiments. D. Dose–dependent inhibition of translation by flavaglines in MDA-MB-231 cells. Compounds were added to cells in culture at the indicated dose for 1 h. ³⁵S-methionine was added to cells 15 min before harvesting, after which TCA precipitable counts were determined and standardized against total protein content. Values are set relative to those obtained from vehicle-treated cells. The values obtained are the average of 2 experiments.

Figure 2. Silvestrol stimulates binding of eIF4A to mRNA.

A. Silvestrol stimulates retention of eIF4AI_f to mRNA. Radiolabeled CAT mRNA was incubated with eIF4AI_f and ATP in the presence of vehicle (DMSO), pateamine A (PatA), hippuristanol , or silvestrol (Sil) for 2 min after which time the reactions were applied to nitrocellulose filters, washed and dried. The amount of radiolabeled mRNA retained on the filters was determined by scintillation counting. The results are the average of three experiments with the standard error of the mean shown. B. Silvestrol stimulates cap-dependent crosslinking of eIF4A_c. Chemical crosslinking of initiation factors to oxidized ³²P-cap labeled CAT mRNA. The presence of 20 µM silvestrol is indicated above the panel. The gel was dried and exposed to X-ray film (Kodak) at −80°C with an intensifying screen. C. Left Panel: Silvestrol reduces the amount of eIF4A_c in the eIF4F complex. RSW was incubated with vehicle (0.5% DMSO) or 50 µM silvestrol for 1 h followed by m⁷GTP-affinity purification. Reactions were resolved on a 10% SDS-polyacrylamide gel followed by Western blot analysis. Right Panel: MDA-MB-231 cells were treated with 25 nM silvestrol for 4 h, after which time, cell extracts were prepared and m⁷GTP-affinity purifications performed. Reactions were resolved on a 10% SDS-polyacrylamide gel followed by Western blot analysis. D. Krebs-2 extracts were treated with 50 µM of silvestrol for 10 min at 30°C. After incubation, poly (rG)-affinity purifications were performed. Eluates were resolved on a 10% SDS polyacrylamide gel and analyzed by Western blotting.

Figure 3. Silvestrol inhibits translation in vivo in MDA-MB-231 breast and PC-3 prostate cancer cells.

A. Relative rate of ³⁵S-Met incorporation in MDA-MB-231 breast and PC-3 prostate cancer cells as a function of silvestrol concentration. Cells were exposed to the indicated concentrations of silvestrol for 1 h in Met-free DMEM with 10% dialyzed FBS, during which the last 15 min, ³⁵S-Met was added. Results are the average of duplicates with the error of the mean shown. Values are standardized against total protein content and plotted relative to DMSO controls, which were ∼100,000 and 400,000 cpm/µg for MDA-MB-231 and PC-3 cells, respectively. B. Kinetics of protein inhibition and cell death following exposure to silvestrol. Cells (60,000 per well in a 24-well plate) were exposed to 25 nM silvestrol for the indicated periods of time. One set of cells was used to quantitate protein synthesis following ³⁵S-Met-labeling, TCA precipitation, and scintillation counting. A parallel set of dishes (200,000 cells per well in a 6-well plate) was used to measure the percentage of viable cells by Annexin V/P.I. staining followed by FACs analysis. These values were normalized to those obtained in the presence of vehicle (1% DMSO), which was set to 100%. C. Reduction in expression of eIF4E-responsive gene products in MDA-MB-231 cells exposed to silvestrol. Western blotting was used to evaluate protein levels from cell lysates harvested at the indicated time points. The panels for survivin (lanes 7 and 8) were analyzed on the same gel, but not on adjacent lanes.

Figure 4. Long-term administration of silvestrol is well tolerated.

A. Silvestrol causes a transient depression of protein synthesis in liver of mice receiving compound. Analysis of liver polysomes from mice injected two consecutive days with 0.2 mg/kg silvestrol and taken 3 or 6 h after the last injection. B. Following 8 consecutive daily administrations of silvestrol (0.2 mg/kg) into Balb/c male mice, bone marrow (BM) and spleen (SP) cell populations were quantitated by FACs analysis. The relative percentage (%) of each population is shown. C. Silvestrol does not alter spleen or liver weights or increase liver aminotransferase activity. Eight Balb/c mice were administered daily injections of vehicle or silvestrol for 8 days. Alanine (ALT) and aspartate (AST) aminotransferase levels, and spleen and liver weights were determined one day after the last injection. The bar represents the mean of the measurements set relative to levels obtained from control mice for each cohort.

Figure 5. Silvestrol inhibits protein synthesis and suppresses endothelial cell tube formation.

A. The relative rate of ³⁵S-Met incorporation in HUVECs as a function of silvestrol concentration. Cells were exposed to the indicated concentrations of silvestrol for 1 h, and in the last 15 min, ³⁵S-Met was added to the cells. Extracts were prepared and the amount of TCA-insoluble ³⁵S-Met determined. Results are the average of duplicates with the error of the mean too small to be seen. Values are standardized against total protein content. B. Kinetics of protein inhibition versus cell death following exposure of HUVECs to silvestrol. HUVECs were exposed to 25 nM silvestrol for the indicated periods of time. One hour before the end of treatment, media was removed, cells washed with PBS and incubated for an additional hour with silvestrol in Met-free DMEM. For the last 15 min, cells were labeled with ³⁵S-Met, followed by TCA precipitation and scintillation counting. A parallel set of dishes (200,000 cells/well in a 6-well plate) were used to measure the percentage of viable cells by Annexin V/P.I. staining and FACs analysis. These values were normalized to those obtained in the presence of 1% DMSO, which was set at 100%. These values are plotted on the right ordinate and as a dashed line. C. Disruption of tube formation by HUVECs in the presence of silvestrol. HUVECs were seeded on BD Matrigel™ Matrix basement membrane (BD Biosciences, Bedford MA) in 24-well plates in triplicate in the presence of increasing concentrations of silvestrol and 24 h later monitored for tube formation. Photographs were taken with a Nikon eclipse TE300 microscope. The bar at the bottom of each photograph corresponds to 50 µm D. Quantitation of tubules (chord-like structures) observed per field. A total of 15 different fields were used for each data point and the errors represent the standard deviation.

Figure 6. Silvestrol suppresses xenograft tumor growth.

A. Response of nude mice bearing early passage human MDA-MB-231 breast cancer to silvestrol. Dosing schedule is indicated at the start of Day 11. Small crosses indicate loss of 2 animals during the course of the experiment. B. Response of nude mice bearing late passage human MDA-MB-231 breast cancer to silvestrol, doxorubicin, or rapamycin. Dosing schedule is indicated at the start of Day 11. C. Xenografts were evaluated by TUNEL and immunohistochemistry for Ki67. Representative data are shown for control-treated and silvestrol-treated mice.