silvestrol and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

Protein synthesis is a powerful therapeutic target in leukemias and other cancers, but few pharmacologically viable agents are available that affect this process directly. The plant-derived agent silvestrol specifically inhibits translation initiation by interfering with eIF4A/mRNA assembly with eIF4F. Silvestrol has potent in vitro and in vivo activity in multiple cancer models including acute lymphoblastic leukemia (ALL) and is under pre-clinical development by the US National Cancer Institute, but no information is available about potential mechanisms of resistance. In a separate report, we showed that intraperitoneal silvestrol is approximately 100% bioavailable systemically, although oral doses were only 1% bioavailable despite an apparent lack of metabolism. To explore mechanisms of silvestrol resistance and the possible role of efflux transporters in silvestrol disposition, we characterized multi-drug resistance transporter expression and function in a silvestrol-resistant ALL cell line generated via culture of the 697 ALL cell line in gradually increasing silvestrol concentrations. This resistant cell line, 697-R, shows significant upregulation of ABCB1 mRNA and P-glycoprotein (Pgp) as well as cross-resistance to known Pgp substrates vincristine and romidepsin. Furthermore, 697-R cells readily efflux the fluorescent Pgp substrate rhodamine 123. This effect is prevented by Pgp inhibitors verapamil and cyclosporin A, as well as siRNA to ABCB1, with concomitant re-sensitization to silvestrol. Together, these data indicate that silvestrol is a substrate of Pgp, a potential obstacle that must be considered in the development of silvestrol for oral delivery or targeting to tumors protected by Pgp overexpression.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Data Interpretation, Statistical; Down-Regulation; Drug Resistance, Neoplasm; Humans; Peptide Chain Initiation, Translational; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Synthesis Inhibitors; RNA, Small Interfering; Triterpenes; Up-Regulation

Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC(50) (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Emu-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.

Topics: Animals; Apoptosis; B-Lymphocytes; Blotting, Western; Chromosome Deletion; Chromosomes, Human, Pair 17; Female; Humans; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Mice; Mice, Inbred C3H; Mice, SCID; Mice, Transgenic; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Biosynthesis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; T-Lymphocytes; Transplantation, Heterologous; Triterpenes