Abstract
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells in the blood, secondary lymphoid tissues, and marrow. The leukemia cells primarily are arrested in the G0/G1 phase of the cell cycle and appear resistant to programmed cell death. Several anti-apoptotic proteins are over expressed in CLL and this correlates with resistance to treatment, disease progression and overall poor prognosis.
Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L), Bcl-2, and Mcl-1, are overexpressed in many cancers including CLL and contribute to tumour initiation, progression and resistance to therapy. Mcl-1 is of particular interest because this molecule appears to be regulated by Nurse-like Cells and other stromal cells that promote survival of CLL cells in vitro and very likely also in vivo.
These proteins enhance the resistance of CLL cells to spontaneous and/or drug-induced apoptosis primarily by interacting with, and antagonizing the activity of mitochondria membrane pro-apoptotic proteins such as Bax and Bak. The protein-protein interaction of Bcl-2 family members is critical for their activity, and these interactions are governed by binding to the BH3 domain. Racemic gossypol is found in cotton seeds and has been studied as a cytotoxic agent in cancer cell lines and in clinical studies in patients with a large variety of cancers. The antitumor activity of racemic gossypol appears to reside principally in the R-(−)-enantiomer (AT101), with reduced activity observed for the S-(+)-enantiomer. AT101 is an antagonist of the BH3-binding groove of the Bcl-2 family of proteins that can inhibit the interactions of these proteins with pro-apoptotic molecules.
We examined whether AT101 could induce apoptosis in Chronic Lymphocytic Leukemia (CLL) and its ability to bind in vitro anti-apoptotic molecules from the Bcl-2 family. Using a Fluorescence Polarization Assay (FPA) we studied the competitive binding affinity of AT101 to Bcl-2 family member proteins. We observed that both, racemic gossypol and AT101 had comparable affinity for Bck-2, Bcl-B, Bfl-1 with EC50=0.6 to 10 μM range. AT101 had a stronger binding affinity to Bcl-X(L) (EC50=0.998 μM vs. 3.084 μM for racemic gossypol), and to Mcl-1 (EC50= 0.52μM vs. 1.07μM for racemic gossypol).
CLL cells were incubated with AT101 for 48 hrs at different concentrations. We observed that leukemia cells were induced to undergo apoptosis in a time and dose dependent manner and that this effect was independent of ZAP-70 expression or IgVH gene mutational status (IC50= 2μM). Cells undergoing apoptosis showed PARP-1 cleavage and upregulation of pro-apoptotic molecules such as Bid, p53, as well as downregulation of Mcl-1.
These results indicate that AT101 has stronger pan-specific binding affinity for Bcl-2 family proteins than racemic gossypol, in particular to Mcl-1 and Bcl-X(L), and that this compound induces apoptosis in CLL B cells independently of ZAP-70 expression or IgVH gene mutational status. Because of these encouraging results a clinical trial using AT101 in CLL patients with high-risk features is currently open at our institution.
In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1.
