The Plant-Derived Agent Silvestrol Has B-Cell Selective Activity In Vitro in Chronic Lymphocytic Leukemia Patient Cells and In Vivo in the Tcl-1 Mouse Model of CLL.

Abstract Chronic Lymphocytic Leukemia (CLL) is an incurable disease with limited therapeutic options, especially for high-risk populations such as the del(17p13) patient subset. Currently available therapies for CLL, even if effective, can have significant detrimental effects on remaining T cells, leaving patients at risk of potentially lethal opportunistic infections. New agents with unique mechanisms of action, independence of key resistance pathways, and selectivity for tumor cells are crucial to make an impact on patient survival. Silvestrol, a structurally unique compound isolated from the plant genus Aglaia, exhibited potent activity against several tumor cell lines and moderate in vivo activity in the P388 mouse leukemia model (J. Org. Chem . 2004, 69:3350; ibid. 69:6156). Based on these results, we tested silvestrol against tumor cells obtained from CLL patients. The LC50 (concentration lethal to 50% of cells relative to untreated control) of silvestrol was 6.5 nM at 72 hours by MTT assay. We performed assays to determine CLL patient cell viability at 72 hours with or without drug washout at various times. In these studies, silvestrol showed up to 50% killing at 72 hours with only a four hour exposure, and reached maximum efficacy with a 24 hour exposure. Silvestrol was similarly effective against cells from CLL patients with or without del(17p13). Furthermore, there was no significant difference in silvestrol-mediated cytotoxicity between lymphoblastic cells with a ten-fold overexpression of Bcl-2 relative to control cells. In MTT assays using isolated CD3+ or CD19+ cells, and in whole blood from healthy volunteers and CLL patients, silvestrol demonstrated substantially more cytotoxicity toward B cells than T cells. We then tested silvestrol using Tcl-1 transgenic mice, which are initially normal but develop a slow-progressing B cell leukemia very similar to human CLL. Lymphocytes obtained from spleens of Tcl-1 mice with leukemia were incubated ex vivo with 80 nM silvestrol and analyzed by flow cytometry. Silvestrol produced an 88% reduction in the B cell percentage after 24 hours with no negative effect on the T cell percentage (8% increase), in contrast to 1 μM fludarabine, which affected both B cell (22% reduction) and T cell (14% reduction) subsets. Non-leukemic mice of the Tcl-1 background strain were treated with 1.0, 1.5 and 2.5 mg/kg/day silvestrol for 5 days to determine a tolerable dose. Three of five mice treated with 2.5 mg/kg/day died at the beginning of the second week of treatment. However, none of the animals treated at 1.0 or 1.5 mg/kg showed signs of toxicity or weight loss even after two full weeks of treatment and were normal at pathological examination. Tcl-1 mice with evidence of leukemia as determined by elevated leukocyte counts and enlarged spleens were then treated with silvestrol at 1.5 mg/kg/day × 5 days for two weeks. Treated mice experienced decreased overall leukocyte counts relative to vehicle controls. Furthermore, CD19+ cell numbers and percentages diminished substantially while the T cells were only mildly affected. Additional leukemic Tcl-1 mice are currently being treated and studies are underway examining the mechanism of action of silvestrol in CLL cells.