Abstract
The apoptotic process and its dysfunctions have become the focus of extensive pharmaceutical research in solid and hematopoietic tumors as well as neurodegenerative diseases. The X-Inhibitor of Apoptosis Protein (XIAP) binds caspase 9, 3 and 7, preventing their activation and, consequently, apoptosis. The Smac/DIABLO protein, released from mitochondria, binds XIAP as a dimer on the same caspase 9 (BIR3 domain) binding site. Similarly, the Smac protein interferes with the XIAP binding site for caspases 3 and 7, thus promoting both the extrinsic and intrinsic apoptotic paths. The thin balance of this binding equilibrium is altered in various tumors, including leukemia, where XIAP is overexpressed and a caspase-dependent resistance to enter apoptosis is usually observed. Thus, XIAP inhibition via Smac mimetics’ binding is at the same time a characterized protein-protein interaction, and a validated mechanism for intervention in cancer therapy. We tested 56 Smac mimetic compounds (designed by CISI - Center for biomolecular Interdisciplinary Studies and Industrial applications of the Milan University) for their in vitro capacity to bind to the XIAP BIR3 domain. We also evaluated the ability of the Smac mimetic compounds to inhibit the growth of the human leukemia HL60, K562 and Jurkat cell lines (derived from patients with promyelocyitic leukemia, blastic phase-CML and T acute lymphoblastic leukemia, respectively). Nine compounds which were shown to be active, were further investigated for their effect on cell cycle (by DNA staining with propidium iodide and cytofluorimetric analysis) and for possible synergistic effect in combination with other chemotherapeutic drugs (Cytarabine, Etoposide and Idarubicine). The same compounds were also tested on normal CD34+ hematopoietic progenitor cells. The cytotoxicity was evaluated after 72 hours treatment with Smac mimetic compounds by a colorimetric assay for the quantification of cell proliferation and viability based on the cleavage of the WST-8 tetrazolium salt by mitochondrial dehydrogenases. The effect of Smac mimetic compounds on CD34+ cells enriched from mobilized peripheral blood was assessed as the capability of inhibiting the myeloid colony growth (CFU-GM). The data were expressed as mean percentage of 3 replicates normalized to the untreated control. Overall, a strong correlation between the binding affinity to the XIAP BIR3 domain and the cytotoxic effect on the leukemic cell lines was observed. The more promising compounds showed IC50 ranging from 0,3 to 1 microM on the HL60 cell line. The Jurkat and K562 cell lines were less sensitive, with IC50 ranging from 11,8 microM to more than 50 microM. However, in the K562 cell line, the combined treatment unveiled synergistic effect with Cytarabine and Etoposide (R Kern index = 1,4 and 1,5 respectively). No cytotoxic effect was observed on normal controls at doses up to 80 microM. A consistent sub G1 apoptotic peak (up to 53% of apoptotic cells) was observed in the HL60 cell line after 48 hrs treatment, thus suggesting a strong activation of the apoptotic process. All together, our data suggest that Smac mimetics may have a promising therapeutic potential as a new class of anticancer drugs in hematopoietic malignancies. Further experiments are currently ongoing to confirm the effectiveness of these compounds also on primary cells from leukemia patients, both as single agents and in combination with conventional drugs. In particular, due to their ability to enhance pro-apoptotic effect, Smac mimetic compounds may allow to overcome resistance of cancer cells to standard chemotherapy.
Improved HDAC Inhibition, Stronger Cytotoxic Effect and Higher Selectivity against Leukemias and Lymphomas of Novel, Tricyclic Vorinostat Analogues