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
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.
- 1. Newman DJ, Cragg GM, Snader KMNatural products as sources of new drugs over the period 1981–2002. J Nat Prod. 2003;66:1022–1037.[PubMed][Google Scholar]
- 2. Butler MSThe role of natural product chemistry in drug discovery. J Nat Prod. 2004;67:2141–2153.[PubMed][Google Scholar]
- 3. Bohnenstengel FI, Steube KG, Meyer C, Nugroho BW, Hung PD, Kiet LC, Proksch PStructure activity relationships of antiproliferative rocaglamide derivatives from Aglaia species (Meliaceae) Z Naturforsch [C] 1999;54:55–60.[PubMed][Google Scholar]
- 4. Proksch P, Edrada R, Ebel R, Bohnenstengel FI, Nugroho BWChemistry and biological activity of rocaglamide derivatives and related compounds in Aglaia species (Meliaceae) Curr Org Chem. 2001;5:923–938.[PubMed][Google Scholar]
- 5. King ML, Chiang CC, Ling HC, Fujita E, Ochiai M, McPhail ATX-ray crystal structure of rocaglamide, a novel antileukemic 1H-cyclopenta[b]benzofuran from Aglaia elliptifolia. J Chem Soc, Chem Comm. 1982:1150–1151.[PubMed][Google Scholar]
- 6. Bohnenstengel FI, Steube KG, Meyer C, Quentmeier H, Nugroho BW, Proksch P1H-cyclopenta[b]benzofuran lignans from Aglaia species inhibit cell proliferation and alter cell cycle distribution in human monocytic leukemia cell lines. Z Naturforsch [C] 1999;54:1075–1083.[PubMed][Google Scholar]
- 7. Baumann B, Bohnenstengel F, Siegmund D, Wajant H, Weber C, Herr I, Debatin KM, Proksch P, Wirth TRocaglamide derivatives are potent inhibitors of NF-kappa B activation in T-cells. J Biol Chem. 2002;277:44791–44800.[PubMed][Google Scholar]
- 8. Fahrig T, Gerlach I, Horvath EA synthetic derivative of the natural product rocaglaol is a potent inhibitor of cytokine-mediated signaling and shows neuroprotective activity in vitro and in animal models of Parkinson’s disease and traumatic brain injury. Mol Pharmacol. 2005;67:1544–1555.[PubMed][Google Scholar]
- 9. Proksch P, Giaisi M, Treiber MK, Palfi K, Merling A, Spring H, Krammer PH, Li-Weber MRocaglamide derivatives are immunosuppressive phytochemicals that target NF-AT activity in T cells. J Immunol. 2005;174:7075–7084.[PubMed][Google Scholar]
- 10. Hwang BY, Su BN, Chai H, Mi Q, Kardono LB, Afriastini JJ, Riswan S, Santarsiero BD, Mesecar AD, Wild R, Fairchild CR, Vite GD, Rose WC, Farnsworth NR, Cordell GA, Pezzuto JM, Swanson SM, Kinghorn ADSilvestrol and episilvestrol, potential anticancer rocaglate derivatives from Aglaia silvestris. J Org Chem. 2004;69:3350–3358.[PubMed][Google Scholar]
- 11. El Sous M, Rizzacasa MABiomimetic synthesis of the novel 1,4-dioxanyloxy fragment of silvestrol and episilvestrol. Tetrahedron Letters. 2005;46:293–295.[PubMed][Google Scholar]
- 12. Nicholson DW, Thornberry NA. Apoptosis. Life and death decisions. Science. 2003;299:214–215.[PubMed]
- 13. Lee SK, Cui B, Mehta RR, Kinghorn AD, Pezzuto JMCytostatic mechanism and antitumor potential of novel 1H-cyclopenta[b]benzofuran lignans isolated from Aglaia elliptica. Chem Biol Interact. 1998;115:215–228.[PubMed][Google Scholar]
- 14. Talanian RV, Quinlan C, Trautz S, Hackett MC, Mankovich JA, Banach D, Ghayur T, Brady KD, Wong WWSubstrate specificities of caspase family proteases. J Biol Chem. 1997;272:9677–9682.[PubMed][Google Scholar]
- 15. Choi C, Kutsch O, Park J, Zhou T, Seol DW, Benveniste ENTumor necrosis factor-related apoptosis-inducing ligand induces caspase-dependent interleukin-8 expression and apoptosis in human astroglioma cells. Mol Cell Biol. 2002;22:724–736.[Google Scholar]
- 16. Ozoren N, Kim K, Burns TF, Dicker DT, Moscioni AD, El-Deiry WSThe caspase 9 inhibitor Z-LEHD-FMK protects human liver cells while permitting death of cancer cells exposed to tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res. 2000;60:6259–6265.[PubMed][Google Scholar]
- 17. Coffey RN, Watson RW, O’Neill AJ, Mc Eleny K, Fitzpatrick JMAndrogen-mediated resistance to apoptosis. Prostate. 2002;53:300–309.[PubMed][Google Scholar]
- 18. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B, Kroemer GSequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med. 1995;182:367–377.[Google Scholar]
- 19. Meurer-Grimes BM, Vairo GL, Yu JTherapeutic compounds and methods. US Patent No. 2003. 6710075.[Google Scholar]
- 20. Hausott B, Greger H, Marian BFlavaglines: a group of efficient growth inhibitors block cell cycle progression and induce apoptosis in colorectal cancer cells. Int J Cancer. 2004;109:933–940.[PubMed][Google Scholar]
- 21. Troy CM, Shelanski MLCaspase-2 redux. Cell Death Differ. 2003;10:101–107.[PubMed][Google Scholar]
- 22. Wang J, Chun HJ, Wong W, Spencer DM, Lenardo MJCaspase-10 is an initiator caspase in death receptor signaling. Proc Natl Acad Sci USA. 2001;98:13884–13888.[Google Scholar]
- 23. Decker P, Muller SModulating poly (ADP-ribose) polymerase activity: potential for the prevention and therapy of pathogenic situations involving DNA damage and oxidative stress. Curr Pharm Biotechnol. 2002;3:275–283.[PubMed][Google Scholar]
- 24. Marcelli M, Cunningham GR, Walkup M, He Z, Sturgis L, Kagan C, Mannucci R, Nicoletti I, Teng B, Denner LSignaling pathway activated during apoptosis of the prostate cancer cell line LNCaP: overexpression of caspase-7 as a new gene therapy strategy for prostate cancer. Cancer Res. 1999;59:382–390.[PubMed][Google Scholar]
- 25. Marcelli M, Cunningham GR, Haidacher SJ, Padayatty SJ, Sturgis L, Kagan C, Denner LCaspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP. Cancer Res. 1998;58:76–83.[PubMed][Google Scholar]
- 26. Mathiasen IS, Jaattela MTriggering caspase-independent cell death to combat cancer. Trends Mol Med. 2002;8:212–220.[PubMed][Google Scholar]
- 27. Chen M, Wang JInitiator caspases in apoptosis signaling pathways. Apoptosis. 2002;7:313–319.[PubMed][Google Scholar]
- 28. Boatright KM, Salvesen GSMechanisms of caspase activation. Curr Opin Cell Biol. 2003;15:725–731.[PubMed][Google Scholar]
- 29. Kischkel FC, Lawrence DA, Tinel A, LeBlanc H, Virmani A, Schow P, Gazdar A, Blenis J, Arnott D, Ashkenazi ADeath receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8. J Biol Chem. 2001;276:46639–46646.[PubMed][Google Scholar]
- 30. Teitz T, Wei T, Valentine MB, Vanin EF, Grenet J, Valentine VA, Behm FG, Look AT, Lahti JM, Kidd VJCaspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nat Med. 2000;6:529–535.[PubMed][Google Scholar]
- 31. Rokhlin OW, Bishop GA, Hostager BS, Waldschmidt TJ, Sidorenko SP, Pavloff N, Kiefer MC, Umansky SR, Glover RA, Cohen MBFas-mediated apoptosis in human prostatic carcinoma cell lines. Cancer Res. 1997;57:1758–1768.[PubMed][Google Scholar]
- 32. Liang Y, Eid MA, Lewis RW, Kumar MVMitochondria from TRAIL-resistant prostate cancer cells are capable of responding to apoptotic stimuli. Cell Signal. 2005;17:243–251.[PubMed][Google Scholar]
- 33. Liang H, Salinas RA, Leal BZ, Kosakowska-Cholody T, Michejda CJ, Waters SJ, Herman TS, Woynarowski JM, Woynarowska BACaspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells. Mol Cancer Ther. 2004;3:1385–1396.[PubMed][Google Scholar]
- 34. Rokhlin OW, Bishop GA, Hostager BS, Waldschmidt TJ, Sidorenko SP, Pavloff N, Kiefer MC, Umansky SR, Glover RA, Cohen MBFas-mediated apoptosis in human prostatic carcinoma cell lines. Cancer Res. 1997;57:1758–1768.[PubMed][Google Scholar]
- 35. Rokhlin OW, Hostager BS, Bishop GA, Sidorenko SP, Glover RA, Gudkov AV, Cohen MBDominant nature of the resistance to Fas- and tumor necrosis factor-alpha-mediated apoptosis in human prostatic carcinoma cell lines. Cancer Res. 1997;57:3941–3943.[PubMed][Google Scholar]