Silvestrol, a Potential Anticancer Rocaglate Derivative from <em>Aglaia foveolata</em>, Induces Apoptosis in LNCaP Cells through the Mitochondrial/Apoptosome Pathway without Activation of Executioner Caspase-3 or -7
Abstract
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 caspase-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% decreased 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.
Natural products have played an important role in cancer chemotherapy by providing several new drugs and lead structures for further development (1, 2). The cyclopenta[b]benzofuran core, found only in plants of the genus Aglaia (Meliaceae), has afforded interesting lead structures due to its unique carbon skeleton and the potent biological activity of some members of this compound class, known also as rocaglate or rocaglamide derivatives (3, 4). In terms of their potential antitumor propensities, cyclopenta[b]benzofurans have been reported to exhibit potent antiproliferative and cytostatic activity against human cancer cell lines (3, 5). They block protein synthesis and induce cell-cycle arrest at the G2/M transition in certain tumor cell lines (6). Furthermore, these compounds inhibit NF-κB activity by blocking inducible NF-κB DNA binding activity and I-κB degradation as well as expression of NF-κB target genes in T-lymphocytes (7). In regard to their NF-κB inhibitory activity, it was demonstrated that a synthetic derivative of rocaglaol is able to reduce tissue inflammation and neuronal cell death by inhibiting NF-κB and AP-1 signaling, resulting in significant neuroprotection in animal models of neurodegeneration (8). In addition, some rocaglamide derivatives have been suggested as a new source of NF-AT specific inhibitors for the treatment of certain inflammatory diseases (9).
In our recent work, the cyclopenta[b]benzofuran, silvestrol, isolated from the fruits and twigs of Aglaia foveolata, has been found to show very potent in vitro cytotoxic activity against several human cancer cell lines (10). Its potency was comparable to that of the well-known anticancer drug, paclitaxel (Taxol). Furthermore, silvestrol exhibited potent inhibitory activity in vivo against several human cancer cells, which were cultivated in hollow fibers, and implanted intraperitoneally in mice (10). The natural product was also active in the P388 murine leukemia model (10). Interestingly, silvestrol possesses an unusual dioxanyloxy group at the C-6 position, which is a major structural difference from other cyclopenta[b]benzofurans, and it was suggested that this pendant group is important for its potent cytotoxic activity (10, 11). Synthesis of this dioxanyloxy substituent has been completed and synthesis of the molecule of silvestrol is underway (11). Since silvestrol is worthy of further investigation as an anticancer drug candidate, a better understanding of its cellular mechanism of action is warranted. Therefore, the present work was carried out to study silvestrol-mediated apoptosis in the LNCaP human prostate carcinoma cell line.
Two apoptosis pathways are relatively well understood at the molecular level. In the intrinsic pathway, apoptotic signaling impacts mitochondria to induce the release of mitochondrial cytochrome c into the cytosol, where it binds to the adaptor protein Apaf-1 (apoptotic protease-activating factor 1) and procaspase-9. These lead to the formation of the apoptosome and subsequent activation of executioner caspases, such as caspase-3 or -7 (12). In the extrinsic pathway, the cell surface death receptor Fas (CD95/Apo-1), a member of the tumor necrosis factor receptor family, is activated by binding of its ligand leading to the formation of the death-inducing-signaling-complex (DISC). DISC formation then triggers the sequential activation of the initiator caspases, caspase-8 or -10, and the executioner caspases, caspase-3 or -7, either directly or through a mitochondrial pathway.
Our results have demonstrated that silvestrol induces apoptosis through the mitochondrial/apoptosome pathway, suggesting that it follows the well-characterized intrinsic pathway. However, silvestrol-mediated apoptosis did not induce the activation of two major executioner caspases, caspases-3 and -7. We also show the contribution of caspase-10, implicating the potential involvement of the extrinsic pathway in silvestrol-induced apoptosis.
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