Silvestrol, a novel rocaglate derivative from Aglaia silvestris with potential anticancer activity, induces apoptosis through the mitochondrial/apoptosome pathway in LNCaP cells

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Cyclopenta[b]benzofurans such as rocaglate and rocaglamide derivatives have been isolated from Aglaia species, and many of these compounds have been noted for their insecticidal activity. More recently, cyclopenta[b]benzofurans have been found to block protein synthesis and to induce cell cycle arrest at the G₂/M transition in certain tumor cell lines. Silvestrol is a novel rocaglate derivate from Aglaia silvestris that exhibited potent in vitro cytotoxic activity comparable to that of paclitaxel. In this study, we focused on the mechanism by which silvestrol induces apoptosis in LNCaP cells. Silvestrol blocked cell cycle progression at the S and G₂/M transitions and induced apoptosis as judged by the TUNEL assay. Silvestrol was found to disrupt mitochondrial trans-membrane potential and to induce cytochrome c release into the cytoplasm. Protein blot analysis indicated that silvestrol produced an increase of Bcl-xL phosphorylation with a concomitant increase of Bak at the protein level. Silvestrol treatment also resulted in PARP cleavage, which was inhibited by the general caspase inhibitor Boc-D-Fmk (50 μM). Protein blots revealed that silvestrol induced the level of cleaved caspase-9 and Apaf-1. Recent studies suggest that Apaf-1 and procaspase-9 assemble into a heptameric wheel-like caspase-activating complex termed the apoptosome. Procapase-9, which is thought to be activated by autoprocessing upon recruitment to the apoptosome, subsequently activates procaspase 3 and 7. However, in this study, no change in either protein level or proteolytic activity of either procaspase-3 or -7 was observed, suggesting that these proteases are not involved in silvestrol-induced apoptosis in LNCaP cells. Silvestrol was further evaluated with an in vivo murine hollow fiber test. Human tumor cells (KB, LNCaP, and Col2) cultivated in hollow fibers and implanted intraperitoneally (ip) and subcutaneously (sc) in immunodeficient mice. The mice were then injected ip with silvestrol daily for 4 days at either 0.625, 1.25, 2.50 or 5.0 mg/kg body weight. At sacrifice, the fibers were retrieved and cell proliferation analyzed by the MTT assay. Inhibition of proliferation was 12 to 63% (ip) and 0 to 27% (sc) for KB cells, 15 to 83% (ip) and 12 to 16% (sc) for LNCaP cells, and 21 to 77% (ip) and 5 to 23% (sc) for Col2 cells. No significant weight loss was observed in any of the mice. Taken together, our data indicate that silvestrol is a potent cytotoxic agent both in vitro and in vivo that induces apoptosis through a mechanism involving the mitochondrial/apoptosome pathway. Supported by NIH grant U19 CA52956.